Compartments for Synthetic Cells: Osmotically Assisted Separation of Oil from Double Emulsions in a Microfluidic Chip

Abstract

Liposomes are used in synthetic biology as cell-like compartments and their microfluidic productiont hrough double emulsions allows for efficient encapsulationo fv arious components. However,r esidual oili nt he membrane remains ac riticalb ottleneck for creating pristine phospholipid bilayers. It has been discovered that osmotically driven shrinkingl eads to detachment of the oil drop. Separation inside am icrofluidic chip has been realizedt oa utomate the procedure, which allows for controlled continuous production of monodisperse liposomes.Giant unilamellar vesicles (GUVs)a re widely used as model membranes to study the biophysicalp roperties of phospholipid bilayers. [1][2][3] In parallel, they attract increasing attention as cell-like compartments in bottom-up synthetic biology,i n which the long-term goal is to build am inimal cell from scratch. [4][5][6][7][8] Upon selecting aG UV productionm ethod for synthetic biology,t he ability to encapsulate variousc omponents is essential. [2] Conventionalm ethods for the productiono fl iposomes comprise gentle hydration, [9,10] swelling on polymer cushions, [11,12] and electroformation. [13,14] These methods are not alwayso ptimal due to the low GUV yield in physiological buffer;p oor encapsulation efficiency; [2,15,16] and, in some cases, harsh conditions to which delicate biomolecules and smaller vesicles are exposed during preparation. [17] Thisi ssue has been addressed by the phase-transfer method, whichi sb ased on preformed water-in-oil (w/o) emulsion droplets crossing a second o/w interface. [18] In recent years, severalo ther,c oncep-tually similar,m ethods have been developed, with the aim of providing higher productivity andb etter control, namely,m icrofluidic jetting, [19] continuous droplet interface crossing encapsulation (cDICE), [20] microfluidic formation of droplet-stabilized vesicles, [21] and microfluidic production of w/o/w double emulsions. [22] The last approach appears to be the least experimentally demandinga nd multiple setups for double emulsion production have been proposed. Microfluidic chips made out of glass [17,22] or polydimethylsiloxane (PDMS), [23][24][25][26] and organic phases,s uch as octanol, [24] chloroform/hexane, [17] and oleic acid, [27] have been used to produce stable double emulsions, which have found attractive applications for synthetic biology, such as the encapsulationo fs maller vesicles, proteins, and DNA. [17,24,28] Another advantage of the double emulsionp rocedure is the virtual absence of losses, with respectt oe ncapsulated solutions,a nd therefore, it is suitable for valuable substratest hat are availablei nl ow quantities.In addition to efficient encapsulation, mimickingn ature requiresapristine bilayer,w hichw ould not compromise membrane-related phenomena, such as the folding of reconstituted membrane proteins.H owever,t he presence of residual oil is an inherentv ice of GUVs prepared from double emulsions, which necessitates removal of the organic phase. So far,afew approaches for solventr emoval have be...

Characterization of lipid composition and diffusivity in OLA generated vesicles

“…Shearing/squeezing, as well as surface tension minimization are believed to be the main drivers of the separation of the double emulsion in OLA [ 14 ]. Krafft et al have recently demonstrated that osmotic shrinking can enhance the separation of oil droplets from GUVs in other microfluidic double emulsion techniques [ 61 ]. This may be used in future studies to investigate whether or not this effect can also be used for improving the separation of the octanol pocket in OLA derived vesicles.…”

Protocells: Milestones and Recent Advances

“…With this design, Deshpande et al [335] used an octanol-assisted system to construct a coacervate-in-liposome system. Krafft et al [347] developed a method to de-wet a vesicle fabricated in a w/o emulsion, using an osmotic gradient composed of sucrose and sodium chloride. Even triple emulsions can be prepared by means of droplet generator microfluidics.…”

Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems?

“…These aqueous phases impede the use of centrifugation as a purification method. As an alternative, Krafft and coworkers developed a simplified system relying solely on sucrose/salt solution as the inner and outer aqueous phases [58]. To completely dewet the Droplet-based microfluidics features various functional units: a droplet production unit to encapsulate a variety of chemical and biochemical contents [26]; a pico-injector for the sequential injection of picoliter volumes into individually produced droplets [94]; a fusion module to provoke the fusion of two distinct droplets, thus enabling the mixture of various chemical and biochemical reagents with high temporal control [95]; a mixing module to improve convection and thereby facilitate molecular reactions within the droplet [96]; a sorting module to precisely separate individual droplets based on various analytical signals such as fluorescence [62] or mass spectrometry [97]; a releasing unit to liberate the entrapped mixture from the droplet into an aqueous continuous phase by applying an electrical field [98] or by using a passive trapping structure [26]; and a detection or observation module that facilitates the assessment of a large droplet population [43,99].…”