ZWITTERIONIC POLYMERS FOR FORMING NANODISCS

CROSS-REFERENCE(S) TO RELATED APPLICATIONS

This application claims priority from United Kingdom patent application number GB2110089.6 filed on 13 July 2021, which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to zwitterionic polymers for solubilising lipid bilayers and forming lipid nanodiscs. In particular it relates to zwitterionic polymers for capturing and isolating membrane proteins in native-like lipid-bilayer nanodiscs.

BACKGROUND TO THE INVENTION

Styrene maleic acid (SMA) polymers are copolymers composed of styrene and maleic acid monomers that can solubilize native lipid:protein complexes directly from cells or raw membranes by forming SMALPs as described in US2012142861 A1. SMALPs are nanoscale disc-shaped lipid assemblies formed from the interaction of membrane lipid bilayers and SMA copolymer as further described in EP1007002B1. Such polymer-stabilized nanodiscs, commonly termed (SMA) lipid particles (SMALPs) use the amphipathic SMA copolymer to wrap around the lipid tails to stabilize the lipids within a nanodisc structure. The SMA copolymer commonly used in SMALP technology has statistically arranged styrene and maleic acid groups which are thought to self-assemble into nanodisc structures by intercalating the planar styrene rings into the lipid tails (perpendicular to the plane of the bilayer) with the maleic acid groups allowing solubilization through hydrogen bonding and ionic interactions with the aqueous solvent.

Accordingly, SMALP technology is a valuable biochemical tool for the solubilization of membrane bilayers and its embedded constituents into nanodiscs without the use of detergents. SMA can extract membrane proteins straight from the native membrane. SMALPs are also effective in structural studies of membrane proteins utilizing techniques such as circular dichroism, analytical ultracentrifugation, electron microscopy, solid state NMR spectroscopy and X-ray crystallography. SMALPs can maintain both the structural stability and function of the encapsulated membrane proteins far more effectively than the detergents which have traditionally been used for the isolation of membrane proteins. Studies have shown that the local environment within a SMALP is very similar in terms of physical properties to the native environment of the membrane proteins. The SMA copolymer, has a maleic acid unit possessing two carboxylic acid groups which undergo partial deprotonation at or around neutral pH. The negatively charged carboxylate groups provides SMA with its hydrophilicity. However, under acidic conditions (pH < 6), both carboxylic acid groups are protonated resulting in the SMA copolymer becoming more hydrophobic and forming a precipitate. Additionally, in the presence of divalent cations at relatively high concentrations (> 5 mM), the two carboxylic acid groups of SMA chelate with the M ²⁺ cation resulting in aggregation. The pH and ionic instability of SMA is problematic when dealing with membrane systems that only solubilize at low pH conditions, or membrane proteins that participate in biochemical reactions that require the presence of divalent metal cations (e.g. Ca ²⁺ , Mg ²⁺ , etc.).

Accordingly, there is a need for polymers with improved pH and ionic stability that are capable of solubilizing membrane systems and their constituents.

The preceding discussion of the background to the invention is intended only to facilitate an understanding of the present invention. It should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was part of the common general knowledge in the art as at the priority date of the application.

SUMMARY OF THE INVENTION

In accordance with an aspect of the invention there is provided a copolymer comprising optionally at least partially substituted styrene monomer units and maleic anhydride derivative monomer units, wherein substantially all the maleic anhydride derivative monomer units include a zwitterionic carboxybetaine moiety.

The copolymer may have the general structure of Formula (I) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

R2 is a branched or linear C1-C6 alkyl;

R ₃ and R ₄ are each independently a branched or linear C ₁ -C ₄ alkyl;

R _a to R _f are each a hydrogen, or at least one of R _a to R _f is independently a methyl, a methoxy, a chloro-, a bromo- or a tert- butyl group and the remaining of R _a to R _t are hydrogen; and

Hal is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

(l -

Ri in the copolymer of Formula (I) may be a C ₂ or C ₃ alkyl. R ₂ in the copolymer of Formula (I) may be a C ₂ or C ₃ alkyl. R ₃ and R4 in the copolymer of Formula (I) may be methyl or ethyl groups. R _a to R _f may each be hydrogen. Hal in the copolymer of Formula (I) may be bromide (Br) or chloride (Cl).

The mole fraction (f) of /V-substituted maleic anhydride derivative monomer units in the copolymer may be between about 0.25 and about 0.5. The number average molecular weight of the copolymer may be equal to or less than 7 kDa. The number average molecular weight of the copolymer may range between about 1.5 kDa and about 5 kDa.

The copolymer may have the structure:

The mole fraction (f) of /V-substituted maleic anhydride derivative monomer units in the copolymer shown immediately above may be between 0.25 and 0.35. The copolymer shown immediately above may have a number average molecular weight of between 3 kDa and 5 kDa. ln accordance with a second aspect of the invention there is provided a lipid nanodisc comprising a lipid bilayer encircled by a copolymer of optionally at least partially substituted styrene monomer units and maleic anhydride derivative monomer units, wherein substantially all the maleic anhydride derivative monomer units include a zwitterionic carboxybetaine moiety, as described above.

The mole fraction (f) of maleic anhydride derivative monomer units in the copolymer of the lipid nanodisc may be between about 0.25 and about 0.5. The copolymer of the lipid nanodisc may have a number average molecular weight that is equal to or less than 7 kDa.

The copolymer encircling the lipid nanodisc may have the general structure of Formula (I)

Formula (I) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

R2 is a branched or linear C1-C6 alkyl;

R3 and R4 are each independently a branched or linear C1 -C4 alkyl; R _a to R _f are each independently a hydrogen, or at least one of R _a to R _f is independently a methyl, a methoxy, a chloro-, a bromo- or a fert-butyl group and the remaining of R _a to Rtare hydrogen; and

Hal is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

(I-)·

The copolymer of the lipid nanodisc may have the following structure: wherein the lipid nanodisc is stable in the presence of multivalent cations or at a pH between 4 and 10. The lipid nanodisc may be stable in the presence of divalent metal cations such as Ca ²⁺ or Mg ²⁺ , particularly at concentrations of the divalent metal cations ranging between 0 and 50 mM. The lipid nanodisc may have a diameter that ranges between about 6 nm and about 20 nm, preferably between about 9 nm and about 11 nm.

In accordance with a third aspect of the invention, there is provided a method of synthesizing a copolymer as described above, the method including the steps of: reacting a precursor copolymer of optionally at least partially substituted styrene monomer units and maleic anhydride monomer units with L/, /V-disubstituted aminoalkyl-1 -amine to form a derivatized copolymer in which the maleic anhydride monomer units have been imidized to N- (/V.AZ-disubstituted amino-alkyl)-maleimide) monomers; and reacting the derivatized copolymer with a w-halo-alkanoic acid to produce the copolymer in which the maleic anhydride derivative monomer units include a zwitterionic carboxybetaine moiety.

To synthesize a copolymer having the structure of Formula (I):

Formula (I) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

R2 is a branched or linear C1-C6 alkyl;

R3 and R4 are each independently a branched or linear C1 -C4 alkyl;

R _a to R _f are each a hydrogen; and

Hal- is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide (I ), the method may include the steps of: reacting poly(styrene-co-maleic anhydride) (SMAnh) copolymer with A/,/V-di(Ci-C4)alkyl- amino(C ₂ -C ₇ )alkyl-1 -amine to form poly(styrene-co-/V-(/V,/V-di(Ci-C4)alkyl-amino-(C2- C ₇ )alkyl)-maleimide); and reacting the poly(styrene-co-/V-(/V,/V-di(Ci-C4)alkyl-amino-(C2-C )alkyl)-maleimide) with a o-halo-(C2-C ₇ )alkanoic acid to produce the copolymer of Formula (I) in which R _a to R _f are each a hydrogen.

The o-halo-(C2-C ₇ )alkanoic acid may be a w-bromo-, w-chloro-, or o-iodo-(C2-C ₇ )alkanoic acid.

The used precursor SMAnh copolymer may have a number average molecular weight equal to or less than 7 kDa. The used precursor SMAnh copolymer may have a styrene-to-maleic anhydride ratio between about 1 :1 and 2.5:1. Accordingly, f in the used SMAnh copolymer may be between about 0.25 and about 0.5.

The poly(styrene-co-maleic anhydride) (SMAnh) copolymer may be reacted with 3 -{N,N- dimethylamino)propyl-1 -amine to form poly(styrene-co-/V-(/V,/V-dimethyl-3-aminopropyl)- maleimide and the poly(styrene-co-A/-(A/’,A/-dimethyl-3-aminopropyl)-maleimi de reacted with 3- bromopropanoic acid to produce a copolymer with the following structure:

In accordance with a fourth aspect of the invention there is provided a method of solubilising a lipid bilayer optionally including one or more membrane proteins, the method comprising mixing the lipid bilayer with an aqueous solution of the above-described copolymer or a copolymer prepared according to the above-described method to produce nanodiscs.

The lipid bilayer may be a cell membrane, optionally including membrane proteins, and the nanodiscs produced may be native nanodiscs.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

Figure 1 is the ¹ H NMR spectrum of R-SMAnh copolymer in DMSO-de; Figure 2 is the ¹³ C NMR spectrum of R-SMAnh copolymer in DMSO-de; Figure 3 is the ATR-FTIR spectra of the SMI2K intermediate (top spectrum) and the butyl carboxylic acid zwitterionic (BCZ2K) copolymer (bottom spectrum);

Figure 4 is the ¹ H NMR spectrum of the BCZ2K copolymer in DMSO-de; Figure 5 is the ¹³ C NMR spectrum of the BCZ2K copolymer in DMSO-de; Figure 6 is the ATR-FTIR spectra of the SMI2K intermediate copolymer (top spectrum) and the propyl carboxylic acid zwitterionic (PCZ2K) copolymer derivative

(bottom spectrum);

Figure 7 is the ¹ FI NMR spectrum of the PCZ2K copolymer in DMSO-de; Figure 8 is the ¹³ C NMR spectrum of the PCZ2K copolymer in DMSO-de; Figure 9 is a graph showing the turbidimetric results of 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) multilamellar vesicles (MLVs) solubilized by a

SMA1000 copolymer and the R-BCZ and R-PCZ copolymers;

Figure 10 is a graph showing the turbidimetric results of DMPC MLVs solubilized by a

SMA1000 copolymer and the BCZ1 K and PCZ1 K copolymers;

Figure 11 is a graph showing the turbidimetric results of DMPC MLVs solubilized by a

SMA2000 copolymer and the BCZ2K and PCZ2K copolymers

Figure 12 is a graph showing the turbidimetric results of DMPC MLVs solubilized by a

SMA copolymer and the PCZ2K copolymer;

Figure 13 is a graph showing the DLS results of DMPC lipids solubilized by a SMA copolymer and the PCZ2K copolymer;

Figure 14 is a graph showing the pH stability results for SMA; Figure 15 is a graph showing the pH stability results for PCZ2K nanodiscs; Figure 16 is a graph showing the ionic stability results for SMA in MgCh (0 - 50 mM);

Figure 17 is a graph showing the ionic stability results for PCZ2K nanodiscs in MgC (0

- 50 mM);

Figure 18 is a graph of the optical density (OD ₆ 2o) measurements of DMPC lipid solubilized by zwitterionic copolymer derivatives and SMA2000 in the presence of 0 - 50 mM MgC ;

Figure 19 is a photograph of the SDS-PAGE gel showing the purification of ZipA protein with zwitterionic and SMA copolymers; and

Figure 20 is a photograph of the SDS-PAGE gel showing the purification of ZipA protein with PCZ2K and SMA copolymers, respectively.

DETAILED DESCRIPTION WITH REFERENCE TO THE DRAWINGS

Zwitterionic copolymers in which the polymer backbone is composed of optionally at least partially substituted styrene monomer units and maleic anhydride derivative monomer units are provided. Substantially all the maleic anhydride monomers in the polymer backbone have been derivatized to include zwitterionic carboxybetaine moieties. As used herein, the phrases “substantially all the maleic anhydride monomers” and “substantially all the maleic anhydride derivative monomer units” refers in each case to at least 90% of the maleic anhydride monomers or maleic anhydride derivative monomer units in the copolymer. The extent of derivatization of the maleic anhydride monomers can be determined by standard techniques for chemical analysis, such as elemental analysis, Fourier-transform infrared spectroscopy and ¹ H nuclear magnetic resonance spectroscopy or a combination of such techniques.

The styrene monomer units may be at least partially substituted and may, for example be a- methylstyrene, p-methylstyrene, p-methoxystyrene, p-chlorostyrene, p-bromostyrene, p-tert- butylstyrene or the like.

The carboxybetaine groups may be ammoniocarboxylates. Ammoniocarboxylates are neutral groups that can be incorporated in a polymer and have a permanently positively charged ammonium cation and a negatively charged carboxylate group which is not adjacent to the cationic site. The pendant zwitterionic ammoniocarboxylate moieties may have the following general structure: The zwitterionic copolymers are polybetaines in which the cations and anions are evenly charged at or around neutral pH conditions. The cationic moiety of the polybetaine is a quaternary ammonium and the anionic moiety is a carboxylate (carboxybetaine). Polybetaines are biocompatible making them suitable for use in membrane protein applications.

The zwitterionic copolymers may have the general structure of Formula (I)

Formula (I) wherein Ri is a branched or linear C2-C7 alkyl;

FI2 is a branched or linear C1-C6 alkyl;

FI ₃ and FU are each independently a branched or linear C ₁ -C ₄ alkyl;

R _a to R _f are each a hydrogen, or at least one of R _a to R _f is independently a methyl, a methoxy, a chloro-, a bromo- or a fert-butyl group and the remaining of R _a to R _f are hydrogen; Hal is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

(I-); f is the mole fraction of /V-substituted maleic anhydride derivative monomer units in the copolymer; and n is the number average degree of polymerization.

Preferably, the styrene monomers are unsubstituted, i.e., R _a to R _f in Formula (I) are each a hydrogen, in which case the zwitterionic copolymers have the general structure of Formula (II), Formula (II) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

R2 is a branched or linear C1-C6 alkyl;

R ₃ and R ₄ are each independently a branched or linear C ₁ -C ₄ alkyl;

Hal is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

(I-); f is the mole fraction of /V-substituted maleic anhydride derivative monomer units in the copolymer; and n is the number average degree of polymerization.

The zwitterionic copolymer may have a pendant dimethylammonioacetate or diethylammonioacetate group bound to the maleic anhydride monomer and accordingly R ₃ and R4 in the copolymer of Formula (I) or (II) may be methyl or ethyl groups, preferably methyl groups. R3 and R4 may also each independently be a methyl or ethyl group. Ri in the copolymer of Formula (I) or (II) may be a C2 or C ₃ alkyl, preferably a C2 alkyl. R ₂ in the copolymer of Formula (I) or (II) may be a C2-C4 alkyl, preferably a C2 or C ₃ alkyl, more preferably a C ₃ alkyl. If, for example, a w- bromoalkanoic or co-chloroalkanoic acid is used to prepare the zwitterionic copolymer of formula (I) or (II), Hal in the copolymer of Formula (I) or (II) may be bromide (Br) or chloride (Cl ).

The zwitterionic copolymers of Formula (II) may be synthesised from low molecular weight (M _w £ 10 kDa) poly(styrene-co-maleic anhydride) (SMAnh) precursors, preferably precursors with a number average molecular weight (M _n ) equal to or less than 7 kDa. When M _n is equal to or less than 7 kDa, n in Formula (II) is equal to or more than 45.

Poly(styrene-co-maleic anhydride) (SMAnh) copolymers exist in various chemical compositions and molecular weights. Most commercial SMAnh polymers are produced using a continuous stirred tank reactor (CSTR) which follows conventional free radical polymerization. The resulting polymers have a relatively large molecular weight distribution (MWD) of D ~ 2. It has been found that the maleic anhydride monomer always occurs as a single unit in the polymer chain, flanked by styrene on either side. Styrene may occur in larger sequences in the polymer chain.

The selected synthetic route toward SMAnh copolymer influences its chemical composition distribution (CCD) and MWD. SMAnh may have various chemical compositions which are expressed as triads. It can be alternating (SMS), semi-alternating (SSM/MSS), or non-alternating (SSS). The precursor SMAnh copolymer may also be a poly(styrene-alt-maleic anhydride) (R- SMAnh) copolymer with a lower MWD (D ~ 1 .4) synthesized via RAFT polymerization. The RAFT end-group may be cleaved off via radical-induced reduction and the resultant R-SMAnh used as a precursor or parent copolymer for producing the zwitterionic copolymers. The R-SMAnh copolymer has an alternating sequence (SMS triad) as confirmed by microstructure analysis of the resonance of the aromatic carbon closest to the polymer backbone and the methylene carbon between the styrene and maleic anhydride monomers on the aliphatic backbone where the resonances appeared at <5 = 137.0 - 140.0 ppm and d = 33.0-37.0 ppm, respectively, on the ¹³ C NMR spectrum of the R-SMAnh copolymer.

The ratio of styrene to maleic acid/maleic anhydride monomers in the precursor SMAnh copolymers may range between 1 :1 and 2.5:1. Accordingly, the ratio of styrene to maleic anhydride derivative monomer units in the zwitterionic copolymers provided may also be between 1 :1 and 2.5:1, and is preferably 2:1. It was found that if the styrene content of the zwitterionic copolymers is high, i.e. above 2.5:1 styrene:maleic anhydride ratio, the zwitterionic copolymers are insoluble in water and thus not suitable for solubilising lipid bilayers. Accordingly, the mole fraction (f) of derivatised maleimide monomer units in the copolymer may range between about 0.25 and about 0.5 (i.e. 0.25 < f < 0.5), and preferably between 0.28 and 0.4 (i.e. 0.28 < f < 0.4) to provide a suitably low molecular weight zwitterionic polymer to produce styrene maleic anhydride derivative lipid particles (SMADLPs) or nanodiscs of a selected size f is even more preferably between 0.25 and 0.35, and most preferably about 0.33.

The molecular weight of the zwitterionic copolymers may range between about 1 .5 kDa and 5 kDa. Accordingly, the number-average degree of polymerization (n in Formula (I) or (II)) may range between 7 and 30.

The zwitterionic copolymers provided may be synthesized by reacting a precursor copolymer of optionally at least partially substituted styrene monomer units and maleic anhydride monomer units with /V,A/-disubstituted aminoalkyl-1 -amine to form a derivatized copolymer in which the maleic anhydride monomer units have been imidized to /V-(A/’,/V’-disubstituted amino-alkyl)- maleimide) monomers; and reacting the derivatized copolymer with a w-halo-alkanoic acid to produce the copolymer in which the maleic anhydride derivative monomer units include a zwitterionic carboxybetaine moiety.

In particular, zwitterionic copolymers having the structure of Formula (I),

Formula (I) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

R2 is a branched or linear C1-C6 alkyl;

R ₃ and R ₄ are each independently a branched or linear C ₁ -C ₄ alkyl; R _a to R _f are each a hydrogen;

FHal is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

O ⁾ ; f is the mole fraction of /V-substituted maleic anhydride derivative monomer units in the copolymer; and n is the number average degree of polymerization, i.e., copolymers of formula (II) above, may be synthesized by first reacting poly(styrene-co-maleic anhydride) (SMAnh) copolymer with N,N- di(Ci-C ₄ )alkyl-amino(C ₂ -C7)alkyl-1-amine to form a styrene maleimide intermediate (SMI), poly(styrene-co-A/-(A/’,A/’-di(CrC4)alkyl-amino-(C2-C7)a lkyl)-maleimide), and then secondly reacting the SMI intermediate with a w-halo-alkanoic acid, preferably a w-bromo-, w-chloro-, or w- iodo-(C2-C ₇ )alkanoic acid, to produce the SMAnh-based zwitterionic copolymer derivatives of Formula (I) in which R _a to R _f are each a hydrogen (i.e. formula (II)) as shown in Scheme 1 . SMAnh SMI Formula (II)

Scheme 1 : Synthetic route toward zwitterionic copolymers of Formula (II) in which Ri to FU and Hal/Hah are as defined with respect to Formula (I) and Ft _a to Fit are each a hydrogen. The modification of the precursor SMAnh copolymer into its zwitterionic derivative occurs in two steps. The first step is the reaction with co-(A/,A/-dialkyl amino)alkyl-1 -amine with the maleic anhydride residues of SMAnh. This reaction can take place over a period of 5 hours in water or in polar organic solvents such as dimethylformamide (DMF), dimethylsulfoxide, butanone, acetone or dioxane at a temperature within the range of 110 - 170 °C. In solvents with a boiling point below the reaction temperature, the reaction needs to be conducted under sufficient pressure to prevent the reaction mixture from boiling. Preferably, the reaction is conducted for 5 hours in DMF at 120 °C or in water at 160 °C, and most preferably in DMF at 120 °C. The second step is the reaction with an w-halo-alkanoic acid, such as 3-bromopropanoic acid. This reaction may be conducted in a polar solvent at a temperature of 50 °C for a period of 20 hours. Preferred solvents for this second step are DMF and water.

The precursor SMAnh copolymer may have a number average molecular weight (M _n ) that is equal to or less than 7 kDa, or that ranges between about 2 kDa and about 5 kDa. The precursor SMAnh copolymer preferably has a styrene-to-maleic anhydride ratio of 2:1 (i.e., f is about 0.33) and a number average molecular weight of about 4 kDA. However, f may also be between 0.25 and 0.35 and the precursor SMAnh copolymer may have a number average molecular weight of between 3 and 5 kDa.

The zwitterionic copolymer may have the following structure: PCZ2K is a zwitterionic copolymer with a permanent quaternary ammonium group as the cationic site and a carboxylate group as the anionic site. The permanent quaternary ammonium group results in the copolymer being stable at higher pH values.

It is preferred for the ratio of styrene to maleic anhydride derivative monomer units in PCZ2K to be 2:1 (i.e. f = -0.33) and the number average molecular weight to be about 4 kDa. However, f may also be between 0.25 and 0.35 and PCZ2K may have a number average molecular weight of between 3 and 5 kDa.

The poly(styrene-co-maleic anhydride) (SMAnh)-based propyl carboxylic zwitterionic (PCZ2K) copolymer may be synthesized from a low number average molecular weight (M _n < 7 kDa), commercially available SMA2000 precursor, with a styrene-to-maleic anhydride ratio of 2:1. The readily reactive maleic anhydride unit may be imidized by treatment with 3-(N,-N- dimethylamino)propyl-1 -amine (DMAPA) to afford a styrene maleimide (SMI) intermediate, poly(styrene-co-A/-(A/’,A/’-dimethyl-3-aminopropyl)-male imide). The resulting tertiary amine functionality on the SMI may be quaternized with 3-bromopropanoic acid to yield a SMAnh-based zwitterionic copolymer with a positively charged quaternary ammonium and negatively charged carboxylate zwitterionic moieties.

The copolymers including a zwitterionic carboxybetaine moiety on the maleic anhydride monomer units may be used to solubilise lipid systems and form nanodiscs (or SMADLPs). The nanodiscs are synthetic structures composed of a section of lipid bilayer, often containing a membrane protein, which is surrounded by an outer rim of a stabilising polymer, i.e. one of the zwitterionic polymers described herein.

The lipid nanodiscs formed with the zwitterionic polymers comprise a lipid bilayer encircled or encased or otherwise formed into a nanodisc by a copolymer of optionally at least partially substituted styrene monomer units and maleic anhydride derivative monomer units. To most effectively solubilize a lipid bilayer into relatively stable lipid nanodiscs in an aqueous medium, the ratio between the optionally at least partially substituted styrene monomer units and the maleic anhydride derivative monomer units in the zwitterionic copolymer may be within the range of 1 :1 to 2.5:1 (i.e. 0.25 < f < 0.5) and the zwitterionic copolymer may have a number average molecular weight that is equal to or less than 7 kDa.

The copolymer encircling the lipid bilayer section in the lipid nanodiscs may have the general structure of Formula (I)

Formula (I) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

Fh is a branched or linear C1-C6 alkyl;

Fh and FU are each independently a branched or linear C1 -C4 alkyl; R _a to R _f are each a hydrogen, or at least one of R _a to R _f is independently a methyl, a methoxy, a chloro-, a bromo- or a tert- butyl group and the remaining of R _a to R _f are hydrogen;

Hal- is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

(I-); f is the mole fraction of /V-substituted maleic anhydride derivative monomer units in the copolymer; and n is the number average degree of polymerization.

Preferably, the polymer encircling the lipid bilayer section in the lipid nanodiscs includes styrene monomers and has the general structure of Formula (II) wherein Ri is a branched or linear C ₂ -C ₇ alkyl;

R2 is a branched or linear C1-C6 alkyl;

R3 and R4 are each independently a branched or linear C1 -C4 alkyl; Hal is a halide anion selected from the group consisting of bromide (Br), chloride (Cl ) and iodide

O ⁾ ; f is the mole fraction of /V-substituted maleic anhydride derivative monomer units in the copolymer; and n is the number average degree of polymerization.

The resultant lipid nanodiscs stabilised by a zwitterionic copolymer of Formula (I) may be stable in an aqueous medium or solution containing multivalent cations. In particular, the resultant lipid nanodiscs may be stable in the presence of divalent metal cations such as Mg ²⁺ and Ca ²⁺ at concentrations at or above about 5 mM.

When the copolymer encasing the lipid nanodisc has the following structure the resultant lipid nanodiscs are stable in an aqueous solution containing multivalent cations. The resultant lipid nanodiscs are also stable in an aqueous solution with a pH between 4 and 10. In particular, the resultant lipid nanodiscs are stable in the presence of divalent metal cations such as Ca ²⁺ and Mg ²⁺ . The lipid nanodiscs are stable at concentrations of the divalent metal cations ranging between 0 and 50 mM. The resultant lipid nanodiscs typically have a diameter that ranges between about 9 - 11 nm. The resultant lipid nanodiscs are of relatively uniform size and most have a diameter of about 10 nm.

The zwitterionic polymers provided may be used to solubilise a lipid bilayer optionally including one or more membrane proteins. The lipid bilayer may be a synthetic bilayer or multilamellar vesicles formed from 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), for example, which may include a membrane protein to be studied or tested. The method of solubilising the lipid bilayer involves mixing the lipid bilayer, optionally containing the one or more membrane proteins, with an aqueous solution of the zwitterionic copolymer to produce lipid nanodiscs. It is thought that the nanodiscs form by self-assembly. The nanodiscs may range in diameter from about 9-25 nm depending on the size, topology and spatial charge distribution on the specific zwitterionic polymer used. During the mixing step, a polymerlipid mass ratio ranging between 1 :1 and 3:1 may be used.

The zwitterionic polymers provided may further be used to solubilize cell membranes which may or may not include membrane proteins. The cell membranes are mixed with the zwitterionic copolymer to produce native nanodiscs. The native nanodiscs comprise a section of biologically intact membrane surrounded by the stabilizing zwitterionic copolymer. The native nanodiscs may range in diameter from about 9-25 nm depending on the size, topology and spatial charge distribution on the specific zwitterionic polymer used. The zwitterionic polymers provided can accordingly be used to isolate and/or purify membrane proteins, particularly integral membrane proteins.

Examples

Synthesis

1. Synthesis of poly(styrene-alt-maleic anhydride) (R-SMAnh) via RAFT polymerization

A typical one-pot synthesis of RAFT-mediated poly(styrene-alt-maleic anhydride) (R-SMAnh) was carried out as follows. Briefly, to a 250 mL two-neck round-bottom flask containing a magnetic stirrer bar, STY (14.5 mL; 126 mmol) and MAnh (12.37 g; 126 mmol) monomers, AIBN (0.3 g; 1.86 mmol) initiator and S-butyl-S’-(l-phenylethyl) trithiocarbonate (BPT) (2.08 g; 7.70 mmol) RAFT agent were added and dissolved in MEK (60 mL) - solvent. The flask was sealed with a rubber stopper and degassed by purging with argon for 30 min. At t = 0 h, a sample was collected and set aside. Subsequently, the flask was submerged into an oil bath pre-heated to 60 °C to initiate the polymerization and the reaction was left to stir for 24 h. Upon completion, another sample was collected, and the polymer conversion was tracked using ¹ H NMR analysis. The polymer was then precipitated in excess ice-cold methanol, filtered gravitationally, and dried under vacuum at 60 °C for 24 h to obtain a yellow powder with a yield of 27.7 g, 98 % monomer conversion.

For RAFT end-group removal, R-SMAnh (10.01 g; 22.0 mmol) was dissolved in 1,4-dioxane (150 mL) in a 250 mL round-bottom flask containing a stirrer bar, followed by the addition of benzoyl peroxide (BPO) (5.08 g; 21.0 mmol). The flask was then sealed with a rubber stopper and degassed with argon for 15 min. Without removing the escape needle the flask was submerged into an oil bath, gradually heated to 82 °C and allowed to stir for 5 h. As the reaction proceeded, the colour of the solution changed from yellow to colourless. The reaction mixture was cooled to RT and the polymer was precipitated in excess ice-cold pentane, filtered, and dried under vacuum at 40 °C for 6 h to obtain a white flaky powder with a yield of 9.8 g.

Figure 1 is the ¹ FI NMR spectrum of R-SMAnh copolymer in DMSO-d6 and Figure 2 is the ¹³ C NMR spectrum of R-SMAnh copolymer in DMSO-d ₆ .

2. Synthesis of poly-(styrene-co-A/-(3-(/\F-/V’-dimethylamino)propyl-/V-ma leimide) (SMI) copolymer

To a 250 mL round-bottom flask containing a magnetic stirrer bar, SMAnh (7.5 g; 37.1 MAnh mmol eq) was dissolved in DMF (100 mL) and the flask was submerged into an oil bath. A mixture of DMAPA (7.5 mL; 59.6 mmol) and DMF (20 mL) was prepared separately and transferred into a dropping funnel. This mixture was subsequently added dropwise into the flask over a period of 30 mins and the contents were stirred for a further 2 h at RT. Upon addition, the anhydride groups reacted rapidly with the primary amine of DMAPA inducing nucleophilic ring opening and formation of amide linkages, the product appearing as an off-white precipitate. After 2 h, the temperature was gradually increased to 120 °C and the reaction was refluxed for 5 h to induce dehydration and ring-closure of the maleimide residues. In the process, the precipitate was re dissolved, and the reddish-brown solution was cooled to RT after the reaction reached completion. The polymer was then precipitated in excess cold hexane and dried under vacuum at 40 °C overnight to obtain a faint yellowish product of 9 g, with a 62 % yield. For complete ring closure, the dry SMI polymer was inserted in an oven and subjected to heating at 120 °C for 5 h under atmospheric pressure. Table 1 shows the properties of the SMAnh variants used as precursors. All of the SMAnh copolymer variants exhibited a similar chemical composition based on ATR-FTIR, ¹ H- and ¹³ C NMR spectroscopic analyses. The SMA1000 and SMA2000 variants comprise similar monomer units, varying only in their molar ratios. Table 2 shows the quantities and reaction conditions employed for the syntheses of three SMI variants.

Table 1 : Properties of SMAnh variants used as precursors.

Table 2: Precursors and reaction conditions used to synthesize SMI copolymers.

3. Synthesis of carboxylic acid zwitterionic copolymer derivatives

Upon treating SMI with a co-halo-alkanoic acid, a nucleophilic substitution (SN2) reaction occurs between the tertiary amine and the alkyl halide. The w-halo-alkanoic acids used in this example include 4-bromobutyric acid (4-BrBC) and 3-bromopropanoic acid (3-BrPC), both of which formed zwitterionic structures with a quaternary ammonium as a permanent cationic site screened by a bromide counter ion as well as anionic sites of carboxylates.

A different solvent system was used for different w-halo-alkanoic acid precursors with amounts and reaction conditions shown in Table 3. The 3-BrPC substituent is used to demonstrate the procedure. In a 100 ml. round-bottom flask equipped with a magnetic stirrer bar, SMI2K (2 g: 1.8 TA mmol eq) was dissolved in DMF (20 ml_). The flask was then submerged into an oil bath pre heated to 50 °C. In a separate vial 3-bromopropanoic acid (1 g; 2.7 mmol) was dissolved in DMF (10 mL) and transferred into a dropping funnel. The acid solution was then added dropwise into the flask over a period of 30 mins and the contents were left to stir for 20 h. Upon completion, the solution was cooled to RT and the polymer was precipitated in excess cold diethyl ether, filtered, and dried under vacuum at RT overnight to obtain a yellowish solid product with a yield > 60 %. SMA2000 SMI2K PCZ2K

Scheme 2: Synthetic route toward zwitterionic polymer PCZ2K.

Table 3: Precursors and reaction conditions used to synthesize the zwitterionic copolymer derivatives.

The zwitterionic copolymers were characterized using ATR-FTIR, ¹ H NMR and ¹³ C NMR spectroscopic analyses. The spectroscopic analysis of BCZ2K is shown in Figures 3 to 5 and the spectroscopic analyses of PCZ2K is shown in Figures 6 to 8.

To ensure that the zwitterionic copolymers are feasible for application on biological systems they must be soluble in aqueous medium. Solubility tests revealed that the zwitterionic derivatives listed in Table 3 are all soluble in distilled water. The lipid solubilization efficiency of the water-soluble zwitterionic copolymer derivatives was evaluated via turbidimetric and DLS analyses. DMPC lipid was used as a mode! membrane system to demonstrate the potential of the zwitterionic copolymers to solubilize MLVs into lipid nanodiscs so that the zwitterionic copolymers may be used for membrane protein solubilization from cultured cells.

For the comparative lipid solubility, pH stability and ionic stability studies, hydrophobic SMAnh variants, SMA1000 and SMA2000 were hydrolysed to the hydrophilic SMA form by refluxing 5 % (w/v) SMAnh suspensions in 1 M KOH at 100 °C overnight (basic hydrolysis), followed by precipitation with dropwise addition of 32 % M HCI solution. The precipitate was then washed with diluted HCI (3 c 25 ml_), filtered and dried under vacuum overnight at ambient temperature to obtain a white powder.

1. Lipid solution preparation

A stock solution of DMPC (Avanti Polar Lipids; 14:0 PC) liposomes was prepared by suspension of dry lipid powder to a concentration of 100 mg/mL in 20 mM Tris-HCI, 200 mM NaCI, pH 8.0, followed by sonication in a bath sonicator for 15 min. This stock solution was stored at -20 °C until use. Working solutions of DMPC multilamellar vesicles (MLVs) were produced by dilution of the 100 mg/mL stock solution in 20 mM Tris-HCI, 200 mM NaCI, pH 8.0 followed by sonication in a bath sonicator for 15 min.

Solutions of SMA variants and synthesized zwitterionic copolymers were prepared by dissolving the polymers to a concentration of 50 mg/mL in 20 mM Tris-HCI, 200 mM NaCI, pH 8.0. A 0.5 mg/mL solution of DMPC MLVs was prepared by dilution of the 100 mg/mL DMPC stock solution in 20 mM Tris-HCI, 200 mM NaCI, pH 8.0, followed by sonication in a bath sonicator for 15 min. The polymer-lipid interaction was monitored by measuring the absorbance of 400 nm light using a MultiskanSky spectrophotometer. A 1 mL quartz cuvette containing 500 mΐ of the 0.5 mg/mL lipid solution was placed in the spectrophotometer and allowed to equilibrate for 1 min before the addition of 500 mΐ of polymer solution. The absorbance of the sample was then followed over a period of 10 min. All measurements were performed at ambient temperature (~ 22 °C).

2. DMPC MLV preparation

To make the vesicle solution, lipids [DMPC] were measured using an analytical balance and suspended in chloroform, with 1 mL chloroform being added per 10 mg of lipid. Chloroform was evaporated from the lipid solution using an air tap. The lipids were resuspended in 50 mM Tris, 150 mM NaCI, pH 7.5 buffer to make a 10 mg/mL solution. The lipid solution was separated into 1 mL aliquots and vesicles were formed by conducting five freeze-thaw cycles using the - 80 °C freezer, followed by a thawing step at 42 °C using a heat block. The vesicle solution was then extruded with 11 passes of the solution through nucleopore track-etched polycarbonate membranes (200 nm). 3. Solubilization protocol

Polymer made up to 5% solution using required buffer was added to equal volume of DM PC at 1.25 mg/ml_ and incubated in cold room overnight. BCZ2K solubilized at pH 5 and PCZ2K at pH 7.5.

4. Dynamic light scattering experiments

DLS experiments were performed using a DynaPro Plate Reader III and DYNAMICS software (Wyatt Technology, Haverhill, UK), using the laser wavelength of 825.4 nm with a detector angle of 150°. Each sample (40 mΐ) was loaded into a 384-well glass bottom SensoPlate (Greiner Bio- One, Germany) in triplicate. Each measurement consisted of 5 scans of 5 s, carried out at 25 °C, with the attenuator position and laser power automatically optimized for size determination (nm).

The hydrodynamic volume and size distribution profiles of the resultant lipid nanodiscs were measured using DLS analysis. The measurements were taken using a ZetaSizer Nano series (Malvern Instruments) at 25 °C in a 1 mL glass cuvette. Data were collected for ~ 30 min, averaged at 15 scans with the calibration time set for 2 min. Five measurements of zero second delay were taken in each run. Size-intensity distributions were generated using the ZetaSizer software version 7.1

DLS analysis was used to determine the size of lipid nanodiscs produced by the zwitterionic copolymers and well-known commercial SMA variants (i.e., SMA1000 and SMA2000). Free DMPC lipid composed mostly of MLVs (~ 1000 nm) was used as a control. The results are in Table 4.

Table 4: Sizes of nanodisc (with standard deviations) formed by zwitterionic copolymers as determined by DLS analysis.

The SMA2000 precursor formed nanodiscs of about 5 nm. However, all its zwitterionic copolymer derivatives formed moderately larger nanodiscs (10 - 20 nm) with broader molar mass distributions. The increased hydrodynamic diameters could be due to the polymer backbone having more STY units accompanied by relatively large zwitterionic pendant groups. The results demonstrate that the size of the resultant lipid nanodiscs can potentially be tuned by changing the zwitterionic pendant group, i.e. lengthening the alkyl chains forming part of the ammoniocarboxylate groups. PCZ2K displayed the best lipid solubilization efficiency due to minimal formation of aggregates

5. Turbidimetric analysis

Turbidimetric analysis was used to evaluate the ability of the synthesized zwitterionic copolymers to solubilize DMPC MLVs. Commercially available SMA variants that were used as precursors to the zwitterionic copolymers served as references. The turbidimetric experiments were conducted at ambient temperature (~ 22 °C), where DMPC lipid appears in a fluid phase. A polymer/lipid ratio (v/v) of 1:1 was used for all turbidimetric experiments. Initially, the absorbance of the free lipid was measured for 1 min before the addition of polymer, which induced an immediate decrease in absorbance value indicative of successful lipid solubilization. Qualitatively, the lipid- polymer interaction was followed visually by observing an apparent change insolution from opaque to transparent.

Both RAFT-made SMA and SMA1000 precursors have a STY: MA ratio of 1:1. The results of the turbidimetric analyses are shown in Figures 9 to 12. The immediate decrease in absorbance after 1 min indicates successful solubilization of the DMPC MLVs by all the zwitterionic copolymers. The lower the absorbance value the greater the solubilization efficiency. It was found that the lipid solubilization efficiency of the zwitterionic derivatives from the RAFT- SMAnh precursor (i.e., R-BCZ and R-PCZ) were lower than the commercial SMA1000 variant as indicated by the greater absorbance values shown in Figure 9. The zwitterionic copolymers synthesized from the SMA1000 precursor (i.e., BCZ1 Kand PCZ1 K) solubilized DMPC MLVs more effectively than their commercial SMA1000 counterpart as is evident from Figure 10. The difference in solubilization efficiency is assumed to be based on the influence of the various anionic groups. Further, the shorter alkyl chain of the propyl carboxylic acid substituent (PCZ1 K) solubilized the DMPC MLVs more efficiently than BCZ1K. The zwitterionic copolymers BCZ2K and PCZ2K were markedly more effective at solubilizing DMPC MLVs than the commercial SMA2000 copolymer as is evident from Figure 11, and it was found that the solubilization efficiency improved when the alkyl chain length of the substituents was shortened from the butyl carboxylic acid (BCZ2K) to propyl carboxylic acid (PCZ2K). This could be attributed to the increased magnitude of the dipole moment and hydrophilicity in the propyl carboxylic acid substituent. The results of the turbidimetric and dynamic light scattering (DLS) analyses demonstrate that the PCZ2K copolymer is capable of solubilizing multilamellar vesicles into nanodiscs of about 10 nm in diameter. Figure 12 shows the turbidimetric analysis results of 1 ,2-dimyristoyl-s/7-glycero-3- phosphocholine (DMPC) lipid solubilized by the SMA copolymer and the PCZ2K copolymer. Figure 13 includes the DLS results of DMPC lipids solubilized by a SMA copolymer and the PCZ2K copolymer. pH stability pH influences the solubilization of lipids and membrane proteins. A low pH environment has a similar effect on SMA as divalent cations, both inducing aggregation although differing in their mechanisms.

The pH stability of nanodiscs produced by the zwitterionic copolymers was evaluated by measuring the change in hydrodynamic diameter and optical density. SMA2000P (Cray Valley) with STY: MAnh ratio of 2:1 (from here on simply referred to as SMA) was used as a reference for the pH stability experiments.

The following buffer systems were prepared for a range of pH values: pH 4 and 5 = Sodium acetate 50 mM, NaCI 150 mM; pH 6 and 7 = KH ₂ PO ₄ /K ₂ HPO ₄ 50 mM, NaCI 150 mM; pH 8 and 9 = Tris/HCI 50 mM, NaCI 150 mM; and pH 10 = CHES/NaOH, NaCI 150 mM.

The zwitterionic copolymer derivatives of Table 3 displayed different pH stability in comparison to SMA as shown in Table 5.

Table 5: Solubility of zwitterionic copolymer derivatives as a function of pH.

^* Sol = soluble; ^** agg = aggregates

SMA copolymer is observed to be most stable at pH 7 but undergoes aggregation at lower pH. Notably aggregation is also observed at extremely basic pH (9 - 10). This would make the SMA polymer more hydrophilic and lessen its lipid solubilization efficiency.

The nanodiscs formed with the PCZ2K copolymer exhibit improved pH stability within the pH range of 4 to 10 in comparison to the nanodiscs formed with SMA copolymer as demonstrated by Figures 14 and 15.

Each zwitterionic copolymer derivative exhibited a unique behaviour at various pH conditions, with only PCZ2K being stable throughout pH 4 - 10. The unique behaviour of the zwitterionic copolymers at various pH conditions demonstrate that the zwitterionic moiety can be tailored to solubilize different types of lipids and membrane proteins.

Ionic stability

The ionic stability of the nanodiscs formed by the zwitterionic copolymers was examined in the presence of MgC at varying concentrations (0 - 50 mM). SMA2000P (simply denoted as SMA) with STY/MAnh ratio of 2:1 was used as a reference. DLS analysis provided the size distribution profiles of all the copolymers. The free SMA copolymer formed nanodiscs of ~ 10 nm. However, in the presence of MgCh at ³ 5 mM concentration aggregation occurred resulting in a dramatic shift and broadening of the size distribution. This is evidence that SMA chelated with the Mg ²⁺ cation resulting in precipitation. The zwitterionic copolymers listed in Table 3 all showed improved stability towards Mg ²⁺ cations compared to SMA. Moreover, the carboxylic acid substituent with a shorter propyl chain (PCZ2K) offers more ionic stability compared to the butyl chain (BCZ2K). Zwitterionic copolymer derivatives from both SMA1000 and RAFT-SMAnh precursors, respectively, exhibited a greater ionic stability compared to SMA. The R-PCZ derivative from the RAFT-SMAnh precursor displayed formation of aggregates in the presence of MgCl2 salt concentration of > 10 mM.

Overall, the zwitterionic copolymers displayed greater tolerance toward Mg ²⁺ cations at higher salt concentration (> 5 mM) compared to commercial SMA. In particular, the nanodiscs formed with the PCZ2K copolymer exhibited improved ionic stability in the presence of MgCH at concentrations of MgCh ranging from 0 to 50 mM as demonstrated by Figures 16 and 17.

To further confirm the ionic stability of the zwitterionic copolymers, light scattering measurements were made after solubilizing DMPC MLVs into nanodiscs in the presence of increasing concentrations of MgC salt. The solubilization of DMPC MLVs into nanodiscs is indicated by the clearing of solutions from opaque to transparent. Therefore, when MgC is present in high concentrations aggregation will occur and the larger aggregates will cause light to scatter. This light scattering was followed by measuring the optical density of the solutions at 620 nm (OD ₆₂ o) using a spectrophotometer. The OD ₆₂ o measurements taken for all copolymers in the presence of 0 - 50 mM MgCl2 are presented in Figure 18.

Model protein purification

A general protein purification protocol was adopted from Lee et al. (Lee, S. C.; Knowles, T. J.; Postis, V. L. G.; Jamshad, M.; Parslow, R. A.; Lin, Y. P.; Goldman, A.; Sridhar, P.; Overduin, M.; Muench, S. P.; Dafforn, T. R. A Method for Detergent- Free Isolation of Membrane Proteins in Their Local Lipid Environment. Nat. Protoc. 2016, 11 (7), 1149-1162.) with minor changes made. The Ni-NTA spin column was equilibrated with 600 mΐ buffer NPI-10 and then centrifuged for 2 min at 890 g (approx. 2900 rpm). Up to 600 mί. of the cleared lysate containing the 6 His- tagged protein was loaded onto the pre- equilibrated Ni-NTA spin column. The Ni-NTA spin column was then centrifuged for 5 min at 270 g (approx. 1600 rpm), and the flow-through was collected and saved for analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The Ni-NTA spin column was then washed twice with 600 mΐ buffer NPI-20, before centrifugation for 2 min at 890 g (approx. 2900 rpm). The protein was eluted twice with 300 mΐ buffer NPI-500, centrifuged for 2 min at 890 g (approx. 2900 rpm), and the eluate was collected.

SDS-PAGE analysis was prepared by adding NuPAGE LDS sample buffer and NuPAGE reducing agent to the previously collected protein eluant samples. Subsequently, a XCell Surelock mini cell was prepared and filled with 1 SDS running buffer. The protein samples were then loaded on the Novex 12 % (w/v) Tris glycine mini gel, along with 10 mί. of HyperPAGE molecular-weight marker in another well for analysis. The tank was attached to a power pack and the gel was run for 35 min at 200 V. The gel was stained with InstantBlue Coomassie stain for 1 h. The gel was destained with ddH20 with gentle shaking at room temperature for 2 - 5 h; the ddH20 was changed a few times during destaining to aid the development of the stain.

The ZipA purification results were obtained from an SDS-PAGE gel that was stained with InstantBlue Coomassie stain. For SMA, a single band is observed around 55 kDa indicating the presence of ZipA protein. The appearance of only a single band is evident that the ZipA protein was isolated in high purity. Moreover, the intensity of the band corresponds to the concentration of the purified ZipA protein.

All zwitterionic copolymers also displayed a single band around 55 kDa, except for BCZ2K as shown in Figure 19. Accordingly, the zwitterionic copolymers of Table 3, except for BCZ2K, isolated the ZipA protein in high purity.

The PCZ2K copolymer isolated the model protein, ZipA, with high purity and at high concentration. Figure 20 is of a photograph of the SDS-PAGE gel showing the ZipA purification results for PCZ2K and SMA copolymers, respectively.

The zwitterionic copolymers described herein have a unique topology compared to carboxybetaine polymers with a different polymer backbone (i.e. not an optionally partially substituted SMAnh backbone) which make them useful for solubilizing lipid assemblies. In particular, the zwitterionic polymers can be applied in so-called SMALP technology that is, among others, used for the isolation of membrane proteins from cell membranes. The zwitterionic polymers have improved ionic stability in comparison to SMA.

Moreover, the zwitterionic polymers described herein are not as cumbersome to prepare as other zwitterionic copolymers used in SMADLP technology.

In particular, PCZ2K has improved stability against increased ionic strength and over a broader pH range than SMA and diisobutylene maleic acid (DIBMA) copolymers. In addition to the improved pH and ionic stability, the PCZ2K copolymer is able to isolate a model protein, i.e., ZipA, with high purity and high concentration, and this behaviour is comparable to commercially available SMA2000P variant with a similar polymer backbone.

The foregoing description has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure. For example, the zwitterionic copolymers provided can be produced from statistical poly(styrene-co-maleic anhydride) (coSMAnh) polymers or from reversible addition-fragmentation chain transfer synthesized copolymers with narrow molecular weight distribution and alternating styrene and maleic acid/maleic anhydride groups with a polystyrene tail, (altSMAnh). Furthermore, should the styrene monomers of the zwitterionic copolymer be at least partially substituted, the copolymer structure (monomer arrangement) and the monomer ratio of optionally substituted styrene monomers relative to the maleic anhydride derivative monomers may vary as may be appropriate for the particular substituted styrene monomer present in the precursor copolymer.

The spacer length between the cationic and anionic sites in the carboxybetaine group may vary, but the cationic and anionic sites should not be too close to one another for the charges of the zwitterionic moieties to permanently interfere with each other. In other words, there should be at least one, but preferably two or more methylene units between the carboxylate group and the ammonium cationic group. The alkyl chain lengths (R groups in Formula (I) and (II)) in the pendant zwitterionic groups on the maleic anhydride monomer units may be tailored to produce lipid nanodiscs of different diameters or with different pH stabilities and/or other desired behaviour or characteristics.

The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims. Finally, throughout the specification and accompanying claims, unless the context requires otherwise, the word ‘comprise’ or variations such as ‘comprises’ or ‘comprising’ will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.