Hierarchical Unilamellar Vesicles of Controlled Compositional Heterogeneity

Hadorn, Maik; Boenzli, Eva; Hotz, Peter Eggenberger; Hanczyc, Martin M.

doi:10.1371/journal.pone.0050156

Cited by 28 publications

(30 citation statements)

References 30 publications

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“…Such level of control of compartment number, content and size could not be achieved using alternative non-microfluidic methods. 124 Vesosomes bear an organisational similarity to multiorganelle eukaryotic cells. Building on this analogy, vesosomes containing a nucleus-like inner compartment were constructed, which contained the components necessary for in vitro transcription of RNA from a DNA template.…”

Section: Vesicles-in-vesiclesmentioning

confidence: 99%

Droplet microfluidics for the construction of compartmentalised model membranes

et al. 2018

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The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.

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Section: Vesicles-in-vesiclesmentioning

confidence: 99%

Droplet microfluidics for the construction of compartmentalised model membranes

et al. 2018

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“…Our method for generating GUVs is based on emulsion transfer in microtiter plates [29,30]. In contrast to common protocols, we both generated and imaged GUVs in glass bottom microtiter plates, enabling highthroughput parameter optimization.…”

Section: Guv Preparationmentioning

confidence: 99%

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

et al. 2018

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Early protocells are commonly assumed to consist of an amphiphilic membrane enclosing an RNAbased self-replicating genetic system and a primitive metabolism without protein enzymes. Thus, protocell evolution must have relied on simple physicochemical self-organization processes within and across such vesicular structures. We investigate freeze-thaw (FT) cycling as a potential environmental driver for the necessary content exchange between vesicles. To this end, we developed a conceptually simple yet statistically powerful high-throughput procedure based on nucleic acidcontaining giant unilamellar vesicles (GUVs) as model protocells. GUVs are formed by emulsion transfer in glass bottom microtiter plates and hence can be manipulated and monitored by fluorescence microscopy without additional pipetting and sample handling steps. This new protocol greatly minimizes artefacts, such as unintended GUV rupture or fusion by shear forces. Using DNAencapsulating phospholipid GUVs fabricated by this method, we quantified the extent of content mixing between GUVs under different FT conditions. We found evidence of nucleic acid exchange in all detected vesicles if fast freezing of GUVs at −80°C is followed by slow thawing at room temperature. In contrast, slow freezing and fast thawing both adversely affected content mixing. Surprisingly, and in contrast to previous reports for FT-induced content mixing, we found that the content is not exchanged through vesicle fusion and fission, but that vesicles largely maintain their membrane identity and even large molecules are exchanged via diffusion across the membranes. Our approach supports efficient screening of prebiotically plausible molecules and environmental conditions, to yield universal mechanistic insights into how cellular life may have emerged.

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“…An innovative method to prepare liposomes, which has attracted attention recently [7][8][9][10][11], is based on the inverted emulsion or phase transfer method. It involves the transfer of water-inoil (w/o) emulsion droplets through an oil-water interface saturated with a phospholipid (PL) monolayer.…”

Section: Introductionmentioning

confidence: 99%

“…This emulsion should have appropriate stability to avoid nucleic acid leakage and a suitable size to serve as the inner leaflet of a liposome. This emulsion is carefully placed onto the interface and either spontaneously or external force, the w/o droplets will cross the interface and pick up a second layer of lipids, forming liposomes [7][8][9][10][11]14,15,[18][19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery

Matos

Miranda

Nuari

et al. 2019

Journal of Drug Targeting

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Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favourable for efficient condensation and charge neutralisation of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterisation of asymmetric liposomes, prepared using water-in-oil (w/o) nanoemulsions of phospholipids (PLs) and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer.Liposomes were prepared by passing PL w/o nanoemulsions through an oil-water interface previously saturated with PLs. We used N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) or N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3phosphocholine (NBD-PC) as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA (pDNA) as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive PLs in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10-15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics. ARTICLE HISTORY

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Hierarchical Unilamellar Vesicles of Controlled Compositional Heterogeneity

Cited by 28 publications

References 30 publications

Droplet microfluidics for the construction of compartmentalised model membranes

Droplet microfluidics for the construction of compartmentalised model membranes

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery

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