Protein capsules

Patent metadata

Field	Value
Publication number	US-11878080-B2
Application number	US-201515508876-A
Country	US
Kind code	B2
Filing date	Sep 4, 2015
Priority date	Sep 4, 2014
Publication date	Jan 23, 2024
Grant date	Jan 23, 2024

Abstract

The present invention provides capsules having a shell of material that comprises an assembly of a protein, and the capsule is optionally provided with a network of material within the shell that is an assembly of the protein. The assembly of the protein is obtained or obtainable by the aggregation of the protein, optionally together with another protein. The assembly is a non-covalent assembly of a protein.

First claim

The invention claimed is:

1. A population of capsules, wherein each capsule comprises a shell of material that comprises of a self-assembly of one or more proteins, wherein the self-assembly of the one or more proteins is an amyloid assembly of the one or more proteins held together by non-covalent interactions between neighboring protein molecules, wherein the population of capsules is formed by a method comprising:
(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby generating in the channel a dispersion of discrete regions of the second phase in the first phase, wherein one of the first or second phase is an aqueous phase and the other phase is a water immiscible phase, and the one of the first and second phase comprises one or more proteins suitable for self-assembly, wherein the one or more proteins is provided in monomeric form and is soluble;
(ii) permitting the one or more proteins to self-assemble at the boundary layer of the discrete regions between the first and second phases under denaturing conditions; and
(iii) optionally collecting the outflow from the channel, thereby obtaining a droplet, which contains a capsule,
and wherein the one or more proteins each comprise 50 or more amino acids,
and wherein the droplets have a size distribution having a relative standard deviation (RSD) of at most 10%, and wherein each capsule has an average diameter in the range of 0.5 μm to 100 μm.

2. The population of capsules of claim 1 , wherein the self-assembly of the one or more proteins is a fibril assembly.

3. The population of capsules of claim 1 , wherein each capsule further comprises a network within the shell that is a self-assembly of one or more proteins, wherein the network is optionally connected to the shell.

4. The population of capsules of claim 3 , wherein the network is an amyloid assembly of the one or more proteins.

5. The population of capsules of claim 1 , wherein the one or more proteins is selected from the group consisting of glucagon, myoglobin, haemoglobin, bovine serum albumin (BSA), ovalbumin, silk, egg yolk, immunoglobulin light chain, immunoglobulin heavy chain, β2-microglobulin, transthyretin, serum AA, apolipoprotein, gelsolin, lysozyme, fibrinogen α-chain, cystatin C, ABriPP, leukocyte chemotactic factor 2, ADanPP, Aβ and Aβ protein precursor (AβPP), prion protein, calcitonin, islet amyloid polypeptide, atrial natriuretic factor, prolactin, insulin, lactadherin, kerato-epithelin, lactoferrin, odontogenic ameloblast-associated protein, semenogelin I, α-S2C and K casein, α-synuclein, polyQ expanded huntingtin, actin, neuroserpin, ferritin, tau, androgen receptor protein, ataxin-1, DRPLA, NAC, atrial natriuretic factor, betabellins 15D and 16D, cytochrome C 552 , methionine aminopeptidase, phosphoglycerate kinase, PI3-SH3, β-lactobgolbulin, monellin, HypF, Human complement receptor, human stefin B, GAG factor, yeast prion Ure2p, herpes simplex virus glycoprotein B, adenovirus fibre, α-lactalbumin, β-lactoglobulin, and yeast protein Sup35.

6. The population of capsules of claim 1 , wherein a self-assembly of the one or more proteins is substantially free of the oligomer form of that protein or proteins.

7. The population of capsules of claim 1 , wherein the shell is a material that is a self-assembly of a plurality of proteins.

8. The population of capsules of claim 1 , wherein at least one capsule in the population of capsules is a first capsule holding a second capsule, and each of the first and second capsules consist of a shell of material that is a self-assembly of one or more proteins.

9. The population of capsules of claim 1 , wherein at least one capsule in the population of capsules holds a component.

10. The population of capsules of claim 7 , further comprising a network within the shell that is a self-assembly of a plurality of proteins, and the shell is a material that is a self-assembly of a plurality of proteins.

11. The population of capsules of claim 1 , wherein the capsule shell has pores, and the pore size is:
(i) at most 0.5 μm; and/or
(ii) at least 0.5 nm.

12. The population of capsules of claim 1 , wherein the water immiscible phase is an oil phase.

13. The population of capsules of claim 1 , wherein the one or more proteins each comprises 100 or more amino acids.

14. The population of capsules of claim 1 , wherein the capsules are a microgel.

15. A method of preparing the population of capsules of claim 1 , the method comprising the steps of:
(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby generating in the channel a dispersion of discrete regions of the second phase in the first phase, wherein one of the first or second phase is an aqueous phase and the other phase is a water immiscible phase, and the one of the first and second phase comprises one or more proteins suitable for self-assembly, wherein the one or more proteins is provided in monomeric form and is soluble;
(ii) permitting the one or more proteins to self-assemble at the boundary layer of the discrete regions between the first and second phases under denaturing conditions; and
(iii) optionally collecting the outflow from the channel, thereby obtaining a droplet, which contains a capsule.

16. The method of claim 15 , wherein the flow rate of the first phase is greater than the flow rate of the second phase.

17. The method of claim 15 , wherein the one or more proteins is provided in an aqueous phase.

18. The method of claim 15 , wherein the concentration of the one or more proteins in the first phase or second phase is from 0.1 to 200 μM.

19. The method of claim 15 , wherein the one or more proteins is provided together with a seed, wherein the seed is an assembly of a plurality of protein molecules.

20. The method of claim 19 , wherein:
(i) the length of the seed is from 10 to 500 nm; and/or
(ii) the seed is provided in a phase with the protein at 5 mole % or less with respect to the concentration of the protein in the phase; and/or
(iii) the seed is provided in a phase with the one or more proteins at from 0.02 to 0.5 μM.

21. The method of claim 15 , wherein the one or more proteins is selected from the group consisting of glucagon, myoglobin, haemoglobin, bovine serum albumin (BSA), ovalbumin, silk, egg yolk, immunoglobulin light chain, immunoglobulin heavy chain, β2-microglobulin, transthyretin, serum AA, apolipoprotein, such as apolipoprotein AI, All and AIV, gelsolin, lysozyme, fibrinogen α-chain, cystatin C, ABriPP, leukocyte chemotactic factor 2, ADanPP, Aβ and Aβ protein precursor (AβPP), prion protein, calcitonin, islet amyloid polypeptide, atrial natriuretic factor, prolactin, insulin, lactadherin, kerato-epithelin, lactoferrin, odontogenic ameloblast-associated protein, semenogelin I, α-S2C and K casein, α-synuclein, polyQ expanded huntingtin, actin, neuroserpin, ferritin, tau, androgen receptor protein, ataxin-1, DRPLA, NAC, atrial natriuretic factor, betabellins 15D and 16D, cytochrome C 552 , methionine aminopeptidase, phosphoglycerate kinase, lysozyme, PI3-SH3, β-lactobgolbulin, monellin, HypF, Human complement receptor, human stefin B, GAG factor, yeast prion Ure2p, herpes simplex virus glycoprotein B, adenovirus fibre, α-lactalbumin, β-lactoglobulin, and yeast protein Sup35, such as a protein selected from the group consisting of lysozyme, glucagon, insulin, myoglobin, haemoglobin, bovine serum albumin (BSA), ovalbumin, and silk.

22. The method of claim 15 , wherei

Assignees

• Cambridge Entpr Ltd
• Harvard College

Inventors

• Knowles Tuomas
• Shimanovich Ulyana
• Dobson Christopher
• Weitz David

Classifications

• A61K9/4825 Primary

  Proteins, e.g. gelatin (gelatin capsule shells with substantial amounts of other macromolecular substances A61K9/4816) · CPC title

• A61K9/4833

  Encapsulating processes; Filling of capsules (mechanical aspects A61J3/07) · CPC title

• A61K9/5169

  Proteins, e.g. albumin, gelatin · CPC title

• B01J13/08

  Simple coacervation, i.e. addition of highly hydrophilic material {(combined with spraying B01J13/043; combined with mechanical division B01J13/04)} · CPC title