Method for preparing a functional synthetic cell in form of a giant unilamellar vesicle

Patent metadata

Field	Value
Publication number	US-11389408-B2
Application number	US-201816623363-A
Country	US
Kind code	B2
Filing date	Jun 6, 2018
Priority date	Jun 16, 2017
Publication date	Jul 19, 2022
Grant date	Jul 19, 2022

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

The invention claimed is:

1. A method for preparing a protocell in the 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,
wherein said polymer shell of the droplet is formed from an amphiphilic copolymer being comprised in the oil phase,
wherein the amphiphilic copolymer comprises at least one hydrophobic block and one hydrophilic block, wherein the at least one hydrophobic block is oriented toward the oil phase and the at least one hydrophilic block is oriented toward the aqueous phase,
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,
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 droplet electro-microfluidics making use of an injector,
wherein the lipid included in the inner space of the droplet is a phospholipid, and
wherein the lipid 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.

2. The method in accordance with claim 1 , wherein the polymer shell of the droplet is made of a diblock copolymer, a triblock copolymer or a statistic copolymer.

3. The method in accordance with claim 2 ,
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 hydrophilic 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 polymer 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.

4. The method in accordance with claim 1 , wherein the lipid is selected from the group consisting of phosphocholine, phosphocholine derivatives, phosphoethanolamine, phosphoethanolamine derivatives, phosphatidylcholine, phosphatidylglycerol, phosphatidylglycerol derivatives and arbitrary combinations of two or more of the aforementioned lipids.

5. The method in accordance with claim 4 , 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.

6. 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 a pico-injector.

7. 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.

8. 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 the form of small or large unilamellar lipid-vesicles.

9. The method in accordance with claim 1 , 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.

10. The method in accordance with claim 1 , 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.

11. 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.

12. 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.

13. The method in accordance with claim 12 , 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.

14. 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.

15. A protocell in the form of a polymer shell-stabilized giant unilamellar vesicle comprising a water-based droplet encapsulated by an outer polymer shell, 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, and wherein the polymer shell of the droplet is made of an amphiphilic copolymer.

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

Assignees

• Max Planck Gesellschaft

Inventors

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

Classifications

• A61K9/1273

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

• A61K9/1277 Primary

  Preparation processes; Proliposomes · 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

• A61K9/1271 Primary

  Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911}) · 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