A Method for Preparing a Functional Synthetic Cell in Form of a Giant Unilamellar Vesicle

Patent metadata

Field	Value
Publication number	US-2020170949-A1
Application number	US-201816623363-A
Country	US
Kind code	A1
Filing date	Jun 6, 2018
Priority date	Jun 16, 2017
Publication date	Jun 4, 2020
Grant date	—

Abstract

The present invention relates to a method for preparing a protocell in form of a giant unilamellar vesicle, which comprises the following steps: a) providing a water-based droplet encapsulated by an outer polymer shell, which borders the inner space of the droplet, wherein the droplet has a maximum dimension of 0.5 μm to 1,000 μm, wherein the inner space of the droplet contains at least one lipid, b) transforming the lipid content of the droplet into a lipid bilayer which is arranged at and covers the inner surface of the polymer shell and oil phase in order to form a polymer shell-stabilized giant unilamellar vesicle, c) optionally incorporating one or more proteins and/or nuclei into the polymer shell-stabilized giant unilamellar vesicle provided in step b) and d) optionally removing the polymer shell and oil phase from the polymer shell-stabilized giant unilamellar vesicle and optionally transferring it from the oil to the water phase.

First claim

1 . A method for preparing a protocell in form of a giant unilamellar vesicle, which comprises the following steps:
a) providing a water-based droplet encapsulated by an outer polymer shell, which borders the inner space of the droplet, wherein the droplet has a maximum dimension of 0.5 μm to 1,000 μm, wherein the inner space of the droplet contains at least one lipid, b) transforming the lipid content of the droplet into a lipid bi-layer which is arranged at and covers the inner surface of the polymer shell and oil phase in order to form a polymer shell-stabilized giant unilamellar vesicle.

2 . The method in accordance with claim 1 , wherein the droplet provided in step a) is spherical and has an outer diameter of 0.5 to 1,000 μm.

3 . (canceled)

4 . The method in accordance with at claim 1 , wherein in step a) a dispersion is provided, in which the droplet is dispersed in an oil-phase, wherein an aqueous phase comprising the at least one lipid is contained in the inner space of the droplet.

5 . The method in accordance with claim 4 , wherein the polymer shell of the droplet is made of a copolymer with a lipophilic or hydrophobic end arranged at the outer side and a hydrophilic end arranged at the inner side of the polymer shell.

6 . The method in accordance with claim 5 , wherein the polymer shell of the droplet is made of a diblock copolymer, a triblock copolymer or a statistic copolymer.

7 . The method in accordance with claim 6 ,
wherein i) the polymer shell of the droplet is made of a diblock copolymer comprising a hydrophobic perfluorinated polymer block arranged at the outer side and a hydro-philic polyether glycol block arranged at the inner side of the polymer shell, or wherein ii) the polymer shell of the droplet is made of a triblock copolymer comprising two hydrophobic perfluorinated polymer end blocks and there between a hydrophilic polyether glycol block, wherein the triblock copolymer is folded so that the hydrophobic perfluorinated polymer blocks are arranged at the outer side and that the hydrophilic polyether glycol block is arranged at the inner side of the polymer shell, or wherein iii) the polymer shell of the droplet is made of a statistic copolymer consisting of a combination of a diblock copolymer comprising a hydrophobic perfluorinated poly-mer block arranged at the outer side and a hydrophilic polyether glycol block arranged at the inner side of the polymer shell and a triblock copolymer comprising two hydrophobic perfluorinated polymer end blocks and there between a hydrophilic polyether glycol block, wherein the triblock copolymer is folded so that the lipophilic perfluorinated polymer blocks are arranged at the outer side and that the hydrophilic polyether glycol block is arranged at the inner side of the polymer shell.

8 - 9 . (canceled)

10 . The method in accordance with claim 1 , wherein the lipid included in the inner space of the droplet is a phospholipid.

11 . The method in accordance with claim 10 , wherein the lipid is selected from the group consisting of phosphocholine, phosphocholine derivatives, phosphoethanolamine, phosphoethanolamine derivatives, phosphatidylcholine, phosphatidylcholine derivatives, phosphatidylglycerol, phosphatidylglycerol derivatives and arbitrary combinations of two or more of the aforementioned lipids.

12 . The method in accordance with claim 11 , wherein the lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid) succinyl], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, L-α-phosphatidylcholine, L-α-phosphatidylglycerol and arbitrary combinations of two or more of the aforementioned lipids.

13 . The method in accordance with claim 1 , wherein the at least one lipid is incorporated into the inner space of the droplet during step a) by droplet generation in a flow-focusing microfluidic device and/or wherein the at least one lipid is incorporated into the inner space of the droplet during step a) by electro-microfluidics making use of an injector, which is preferably a pico-injector.

14 . The method in accordance with claim 1 , wherein the at least one lipid is incorporated into the inner space of the droplet during step a) by techniques for water-in-oil emulsion formation such as vortexing or sonication.

15 . The method in accordance with claim 1 , wherein i) the at least one lipid is incorporated into the inner space of the droplet during step a) in form of small or large unilamellar lipid-vesicles.

16 . The method in accordance with claim 1 , wherein the at content of the droplet is transformed during step b) into a lipid bilayer by adjusting the concentration of ions within the inner space of the droplet and/or applying electric fields.

17 . The method in accordance with claim 16 , wherein the ions are magnesium ions and the concentration of magnesium ions within the inner space of the droplet is adjusted by incorporating the at least one lipid into the inner space of the droplet during step a) by droplet generation in a flow-focusing microfluidic device, wherein the lipid containing aqueous phase used therefore has a magnesium ion concentration of 1 to 100 mM, preferably of 2 to 100 mM, more preferably of 5 to 50 mM, still more preferably of 5 to 20 mM and most preferably of 8 to 12 mM, such as in particular of 10 mM.

18 . The method in accordance with claim 16 , wherein the ions are magnesium ions and the concentration of magnesium ions within the inner space of the droplet is adjusted during step b) by electro-microfluidics making use of an injector, which is preferably a pico-injector.

19 . The method in accordance with claim 1 , wherein step c) is performed by incorporating one or more proteins into the polymer shell-stabilized giant unilamellar vesicle provided in step b) by electro-microfluidics making use of an injector, which is preferably a pico-injector.

20 . (canceled)

21 . The method in accordance with claim 1 , wherein during step c) a transmembrane protein and/or a cytoskeleton protein is incorporated into the lipid bilayer and/or into the inner space of the polymer shell-stabilized giant unilamellar vesicle.

22 . The method in accordance with claim, wherein a protein selected from the group consisting of receptors, ATP-synthase, polymerase, actin, tubulin, antibodies, integrins, nuclei as isolated from cells and arbitrary combinations of two or more of the aforementioned proteins and nuclei and arbitrary combinations of two or more of the aforementioned proteins and nuclei are used.

23 . The method in accordance with claim 1 , wherein during step d) the polymer shell and the oil phase are removed from the polymer shell-stabilized giant unilamellar vesicle.

24 . A protocell in form of a polymer shell-stabilized giant unilamellar vesicle comprising an outer polymer shell, which borders an inner space, wherein the giant unilamellar vesicle has a maximum dimension of 0.5 μm to 1,000 μm, and further comprising a lipid bilayer being composed of at least one lipid, wherein the lipid bilayer is arranged at and covers the inner surface of the polymer shell.

25 . A protocell in form of a giant unilamellar vesicle obtainable with a process for preparing a protocell in form of a

Assignees

• Max Planck Gesellschaft

Inventors

• Spatz Joachim P
• Benk Lucia T
• Frohnmayer Johannes Patrick
• Haller Barbara
• Janiesch Jan-Willi
• Plazman Yilia
• Weiss Marian

Classifications

• A61K9/1277 Primary

  Preparation processes; Proliposomes · CPC title

• A61K9/1273

  Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances · CPC title

• A61K9/1075

  Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers (A61K9/0026 takes precedence) · CPC title

• A61K9/0019

  Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00) · CPC title

• A61K47/34

  Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers (A61K47/10 takes precedence) · CPC title