Polymer-based lipid nanodiscs and macrodiscs

Patent metadata

Field	Value
Publication number	US-11092605-B2
Application number	US-201816198397-A
Country	US
Kind code	B2
Filing date	Nov 21, 2018
Priority date	Nov 22, 2017
Publication date	Aug 17, 2021
Grant date	Aug 17, 2021

Abstract

The disclosure provides a lipid nanodisc including a lipid bilayer having two opposing hydrophilic faces and a hydrophobic edge between the hydrophilic faces, and a copolymer encircling the hydrophobic edge of the lipid bilayer, the copolymer including a first monomeric unit including a pendant aromatic group, and a second monomeric unit including a pendant hydrophilic group, wherein the first monomeric unit and the second monomeric unit are present in the copolymer is a molar ratio ranging from 1:1 to 3:1 for the first monomeric unit:the second monomeric unit. The disclosure further provides a method of making the polymer-based lipid nanodiscs of the disclosure and methods of characterizing membrane proteins using the polymer-based lipid nanodiscs of the disclosure.

First claim

What is claimed:

1. A lipid nanodisc comprising:
a lipid bilayer comprising two opposing hydrophilic faces and a hydrophobic edge between the hydrophilic faces; and
a copolymer encircling the hydrophobic edge of the lipid bilayer, the copolymer comprising
a first monomeric unit comprising a pendant aromatic group, and
a second monomeric unit comprising a pendant hydrophilic group,
wherein the first monomeric unit and the second monomeric unit are present in the copolymer in a molar ratio ranging from 1:1 to 3:1 for the first monomeric unit:the second monomeric unit, wherein the copolymer is not zwitterionic.

2. The lipid nanodisc of claim 1 , wherein the first monomeric unit comprises a styrene monomer unit.

3. The lipid nanodisc of claim 1 , wherein the second monomeric unit comprises a modified maleic anhydride or modified maleic acid monomer unit.

4. The lipid nanodisc of claim 1 , wherein the pendant hydrophilic group comprises at least one of hydroxyl, amino, carboxylic acid, carboxylate, phosphate, phosphonate, carboxylic ether, carboxylic ester, phosphate ester, amide, phosphonamide, or salts of the foregoing.

5. The lipid nanodisc of claim 1 , wherein the pendant hydrophilic group comprises at least one of a fluorescent group and a chelating group further comprising a metal ion bound thereto.

6. The lipid nanodisc of claim 1 , wherein the copolymer has a number-average molecular weight ranging from 1 kDa to 6 kDa.

7. The lipid nanodisc of claim 1 , wherein the lipid comprises at least one of phosphatidylethanolamines, phosphatidylcholines, phosphatidylglycerols, phosphatidylserines, cholesterols, sphingomyelin, gangliosides, lipopolysaccharides, phosphatidylinositols, and derivatives of the foregoing.

8. The lipid nanodisc of claim 1 , wherein the nanodisc has a diameter in a range of about 6 nm to about 100 nm.

9. The lipid nanodisc of claim 1 , further comprising a membrane protein spanning across the lipid bilayer from the first hydrophilic face to the second hydrophilic face.

10. The lipid nanodisc of claim 1 , wherein the molar ratio of the pendant hydrophilic group to the second monomer unit in the copolymer is about 2:1 to 1:10.

11. A method of making a lipid nanodisc, the method comprising:
contacting:
a lipid; and
a copolymer comprising
a first monomeric unit comprising a pendant aromatic group, and
a second monomeric unit comprising a pendant hydrophilic group,

wherein the first monomeric unit and the second monomeric unit are present in the copolymer in a molar ratio ranging from 1:1 to 3:1 for the first monomeric unit:the second monomeric unit and the copolymer is not zwitterionic,

to form a lipid nanodisc comprising a lipid bilayer comprising two opposing hydrophilic faces and a hydrophobic edge between the hydrophilic faces and the copolymer encircling the hydrophobic edge of the lipid bilayer.

12. The method of claim 11 , further comprising preparing an aqueous solution of the copolymer prior to contacting the copolymer with the lipid.

13. The method of claim 11 , further comprising preparing a lipid dispersion prior to contacting the copolymer with the lipid.

14. The method of claim 11 , wherein the contacting comprises admixing the lipid and the copolymer in solution.

15. The method of claim 11 , wherein the lipid and the copolymer are provided in a ratio of about 3:1 to about 1:3, by weight.

16. The method of claim 11 , wherein the lipid comprises at least one of phosphatidylethanolamines, phosphatidylcholines, phosphatidylglycerols, phosphatidylserines, cholesterols, sphingomyelin, gangliosides, lipopolysaccharides, phosphatidylinositols, and derivatives of the foregoing.

17. A method of characterizing a membrane protein, the method comprising:
contacting the lipid nanodisc of claim 1 with a membrane protein to form a membrane protein-nanodisc comprising the membrane protein spanning across the lipid bilayer from the first hydrophilic face to the second hydrophilic face; and
characterizing the lipid nanodisc comprising the membrane protein.

18. The method of claim 17 , wherein characterizing comprises at least one of structural characterization and functional characterization, the characterization comprising performing at least one of solution nuclear magnetic resonance (NMR), solid state NMR, circular dichroism, electron paramagnetic resonance (EPR), Fourier transform infrared spectroscopy (FTIR), resonance Raman spectroscopy, ultraviolet-visible spectroscopy (UV/vis), cryo-electron microscopy (cryo-EM), surface plasmon Raman spectroscopy, sum frequency generation (SFG), fluorescence, small angle x-ray scattering (SAXS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and an enzymatic assay.

19. The lipid nanodisc of claim 1 , wherein the lipid nanodisc is prepared by contacting the lipid and the copolymer in a ratio of about 3:1 to about 1:1, by weight.

20. The lipid nanodisc of claim 1 , wherein the nanodisc has a diameter in a range of about 10 nm to about 90 nm.

21. The lipid nanodisc of claim 1 , wherein the copolymer has a number-average molecular weight ranging from 1 kDa to 3 kDa.

22. The lipid nanodisc of claim 10 , wherein the molar ratio of the pendant hydrophilic group to the second monomer unit in the copolymer is about 2:1 to about 1:4.

23. The lipid nanodisc of claim 10 , wherein the molar ratio of the pendant hydrophilic group to the second monomer unit in the copolymer is about 1:1.

Assignees

• Univ Michigan Regents

Inventors

• Ramamoorthy Ayyalusamy
• Ravula Thirupathi
• Hardin Nathaniel Z
• Cox Sarah J

Classifications

• C08F8/12 Primary

  Hydrolysis · 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

• G01N33/6842 Primary

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• C08F8/32

  by reaction with amines · CPC title

• C08F8/44

  Preparation of metal salts or ammonium salts · CPC title