Surfactant-free production of biomimetic artificial cells using PDMS-based microfluidics

AbstractMicrofluidic-based production of cellular mimics (e.g. giant vesicles) presents a paradigm-shift in the development of artificial cells. While encapsulation rates are high and vesicles are mono-disperse compared to swelling-based techniques, current microfluidic emulsion-based methods heavily rely on the addition of additives such as surfactants, glycerol and even ethanol to produce stable vesicles. In this work, we present a microfluidic platform designed for the production of cellular mimics in the form of giant unilamellar vesicles (GUVs). Our PDMS-based device comprises a double cross-junction and a serpentine-shaped shear inducing module to produce surfactant-free and additive-free monodisperse biomimetic GUVs. Vesicles can be made with neutral and charged lipids in physiological buffers and, unlike previous works, it is possible to produce them with pure water both inside and outside. By not employing surfactants such as block co-polymers, additives like glycerol, and long-chain poly-vinyl alcohol that are known to alter the properties of lipid membranes, the vesicles are rendered truly biomimetic. The membrane functionality and stability are validated by lipid diffusion, membrane protein incorporation, and leakage assays. To demonstrate the usability of the GUVs using this method, various macromolecules such as DNA, smaller liposomes, mammalian cells and even microspheres are encapsulated within the GUVs.

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Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics

Communications Chemistry ◽

10.1038/s42004-021-00530-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Naresh Yandrapalli ◽

Julien Petit ◽

Oliver Bäumchen ◽

Tom Robinson

Keyword(s):

High Throughput ◽

Paradigm Shift ◽

Mammalian Cells ◽

Lipid Vesicles ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Artificial Cells ◽

Membrane Function ◽

Lipid Diffusion ◽

Protein Incorporation

AbstractMicrofluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

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Motion of Particles Attached to Giant Vesicles: Falling Ball Viscosimetry and Elasticity Measurements on Lipid Membranes

Perspectives in Supramolecular Chemistry ◽

10.1002/9780470511534.ch15 ◽

2007 ◽

pp. 221-229 ◽

Cited By ~ 1

Author(s):

Rumiana Dimova ◽

Christian Dietrich ◽

Bernard Pouligny

Keyword(s):

Lipid Membranes ◽

Giant Vesicles ◽

Elasticity Measurements

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Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells

Nanomaterials ◽

10.3390/nano9071025 ◽

2019 ◽

Vol 9 (7) ◽

pp. 1025 ◽

Cited By ~ 5

Author(s):

Rejhana Kolašinac ◽

Sebastian Jaksch ◽

Georg Dreissen ◽

Andrea Braeutigam ◽

Rudolf Merkel ◽

...

Keyword(s):

Lipid Membranes ◽

Mammalian Cells ◽

High Efficiency ◽

Phase State ◽

Chinese Hamster ◽

Cationic Lipid ◽

Cationic Liposomes ◽

Biological Macromolecules ◽

Lipid Mixture ◽

Fusion Efficiency

Lipid-based nanoparticles, also called vesicles or liposomes, can be used as carriers for drugs or many types of biological macromolecules, including DNA and proteins. Efficiency and speed of cargo delivery are especially high for carrier vesicles that fuse with the cellular plasma membrane. This occurs for lipid mixture containing equal amounts of the cationic lipid DOTAP and a neutral lipid with an additional few percents of an aromatic substance. The fusion ability of such particles depends on lipid composition with phosphoethanolamine (PE) lipids favoring fusion and phosphatidyl-choline (PC) lipids endocytosis. Here, we examined the effects of temperature, ionic strength, osmolality, and pH on fusion efficiency of cationic liposomes with Chinese hamster ovary (CHO) cells. The phase state of liposomes was analyzed by small angle neutron scattering (SANS). Our results showed that PC containing lipid membranes were organized in the lamellar phase. Here, fusion efficiency depended on buffer conditions and remained vanishingly small at physiological conditions. In contrast, SANS indicated the coexistence of very small (~50 nm) objects with larger, most likely lamellar structures for PE containing lipid particles. The fusion of such particles to cell membranes occurred with very high efficiency at all buffer conditions. We hypothesize that the altered phase state resulted in a highly reduced energetic barrier against fusion.

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ELECTROFUSION OF MODEL LIPID MEMBRANES VIEWED WITH HIGH TEMPORAL RESOLUTION

Biophysical Reviews and Letters ◽

10.1142/s179304800600032x ◽

2006 ◽

Vol 01 (04) ◽

pp. 387-400 ◽

Cited By ~ 20

Author(s):

KARIN. A. RISKE ◽

NATALYA BEZLYEPKINA ◽

REINHARD LIPOWSKY ◽

RUMIANA DIMOVA

Keyword(s):

Electric Fields ◽

Temporal Resolution ◽

Lipid Membranes ◽

Cell Biology ◽

Digital Camera ◽

Extensive Study ◽

High Temporal Resolution ◽

Giant Vesicles ◽

Model Lipid Membranes ◽

Millisecond Time Scale

The interaction of electric fields with lipid membranes and cells has been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest because of their widespread use in cell biology and biotechnology. Giant vesicles, being of cell size and convenient for microscopy observations, are the simplest model of the cell membrane. However, optical microscopy observation of effects caused by electric DC pulses on giant vesicles is difficult because of the short duration of the pulse. Recently this difficulty has been overcome in our lab. Using a digital camera with high temporal resolution, we were able to access vesicle fusion dynamics on a sub-millisecond time scale. In this report, we present some observations on electrodeformation and –poration of single vesicles followed by an extensive study on the electrofusion of vesicle couples. Finally, we suggest an attractive approach for creating multidomain vesicles using electrofusion and present some preliminary results on the effect of membrane stiffness on the fusion dynamics.

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Thermal fluctuations of giant vesicles and elastic properties of bilayer lipid membranes. The role of the excess surface

Progress in Colloid & Polymer Science - Trends in Colloid and Interface Science III ◽

10.1007/bfb0116179 ◽

2007 ◽

pp. 11-17 ◽

Cited By ~ 4

Author(s):

J. F. Faucon ◽

. Méléard ◽

M. D. Mitov ◽

I. Bivas ◽

P. Bothorel

Keyword(s):

Elastic Properties ◽

Lipid Membranes ◽

Thermal Fluctuations ◽

Bilayer Lipid Membranes ◽

Giant Vesicles ◽

Bilayer Lipid ◽

Excess Surface

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Artificial cells drive neural differentiation

Science Advances ◽

10.1126/sciadv.abb4920 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabb4920 ◽

Cited By ~ 4

Author(s):

Ö. Duhan Toparlak ◽

Jacopo Zasso ◽

Simone Bridi ◽

Mauro Dalla Serra ◽

Paolo Macchi ◽

...

Keyword(s):

Mammalian Cells ◽

Artificial Cells ◽

Human Embryonic Kidney Cells ◽

Drug Delivery Vehicles ◽

Physiological Conditions ◽

Therapeutic Molecules ◽

Delivery Vehicles ◽

Smart Drug ◽

Smart Drug Delivery

We report the construction of artificial cells that chemically communicate with mammalian cells under physiological conditions. The artificial cells respond to the presence of a small molecule in the environment by synthesizing and releasing a potent protein signal, brain-derived neurotrophic factor. Genetically controlled artificial cells communicate with engineered human embryonic kidney cells and murine neural stem cells. The data suggest that artificial cells are a versatile chassis for the in situ synthesis and on-demand release of chemical signals that elicit desired phenotypic changes of eukaryotic cells, including neuronal differentiation. In the future, artificial cells could be engineered to go beyond the capabilities of typical smart drug delivery vehicles by synthesizing and delivering specific therapeutic molecules tailored to distinct physiological conditions.

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Psychrotolerant Paenibacillus tundrae Isolates from Barley Grains Produce New Cereulide-Like Depsipeptides (Paenilide and Homopaenilide) That Are Highly Toxic to Mammalian Cells

Applied and Environmental Microbiology ◽

10.1128/aem.00049-12 ◽

2012 ◽

Vol 78 (10) ◽

pp. 3732-3743 ◽

Cited By ~ 17

Author(s):

Stiina Rasimus ◽

Raimo Mikkola ◽

Maria A. Andersson ◽

Vera V. Teplova ◽

Natalia Venediktova ◽

...

Keyword(s):

Lipid Membranes ◽

Mammalian Cells ◽

High Performance ◽

Food Poisoning ◽

Respiratory Control ◽

Structural Difference ◽

Glucose Consumption ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Content Type

ABSTRACTPaenilide is a novel, heat-stable peptide toxin fromPaenibacillus tundrae, which colonizes barley.P. tundraeproduced 20 to 50 ng of the toxin mg−1of cells (wet weight) throughout a range of growth temperatures from +5°C to +28°C. Paenilide consisted of two substances of 1,152 Da and 1,166 Da, with masses and tandem mass spectra identical to those of cereulide and a cereulide homolog, respectively, produced byBacillus cereusNS-58. The two components of paenilide were separated from those of cereulide by high-performance liquid chromatography (HPLC), showing a structural difference suggesting the replacement ofO-Leu (cereulide) byO-Ile (paenilide). The exposure of porcine spermatozoa and kidney tubular epithelial (PK-15) cells to subnanomolar concentrations of paenilide resulted in inhibited motility, the depolarization of mitochondria, excessive glucose consumption, and metabolic acidosis. Paenilide was similar to cereulide in eight different toxicity endpoints with porcine and murine cells. In isolated rat liver mitochondria, nanomolar concentrations of paenilide collapsed respiratory control, zeroed the mitochondrial membrane potential, and induced swelling. The toxic effect of paenilide depended on its high lipophilicity and activity as a high-affinity potassium ion carrier. Similar to cereulide, paenilide formed lipocations, i.e., lipophilic cationic compounds, with K+ions already at 4 mM [K+], rendering lipid membranes electroconductive. Paenilide-producingP. tundraewas negative in a PCR assay with primers specific for thecesBgene, indicating that paenilide was not a product of plasmid pCER270, encoding the biosynthesis of cereulide inB. cereus. Paenilide represents the first potassium ionophoric compound described forPaenibacillus. The findings in this paper indicate that paenilide fromP. tundraeis a potential food-poisoning agent.

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