Vinyl Ether Maleic Anhydride Polymer Lipid Nanodiscs Functionalized with Alanine Enantiomers

Materials

All materials used were obtained from commercial sellers including Sigma-Aldrich and Avanti Polar Lipids, and stored at the appropriate listed conditions unless stated otherwise. All materials used for polymerization were obtained and utilized in a solid state, except for butyl-vinyl ether (BVE), dodecyl-vinyl ether (DVE), and dioxane. The reversible addition-fragmentation chain transfer polymerization (RAFT) chain transfer agent (CTA) used was 2-(propionic acid)yl dodecyl trithiocarbonate (PADTC), and was synthesized as outlined in previous literature3 and stored in a refrigerator kept at approximately 4oC. 

Synthesis of Vinyl Ether Maleic Anhydride (VEMA) Copolymers

Two distinct VEMA copolymers were synthesized and studied. The first VEMA copolymer synthesized (copolymer A) was done so by combining monomers BVE (3.0000g), maleic anhydride (MAn) (2.7709g), and DVE (1.5602g), then mixing with PADTC (0.7926g) and initiator 2,2’-Azobis(isobutyronitrile) (AIBN) (.0829g) in a 50mL round-bottom flask with a stir bar. The molar ratio utilized for each reactant for copolymer A was 13.25 : 12.5 : 3.25 : 1 : 0.15 respectively. The mixture was then dissolved in dioxane (1:1 by mass) (8.2065g). 

The second VEMA copolymer synthesized (copolymer B) was done so by combining monomers BVE (4.5000g) and MAn (3.3043g), then mixing with PADTC (0.9845g) and AIBN (0.2058g) in a 50mL round-bottom flask with a stir-bar. The molar ratio utilized for each reactant for copolymer B was 16 : 12 : 1.0 : 0.3 respectively. The mixture was then dissolved in dioxane (1:1 by mass) (8.9946g). 

Once a solution was obtained for each copolymer, samples were collected for proton 1H NMR analysis in order to verify the presence of all monomers prior to the synthesis reaction. Each 50mL round-bottom flask was then sealed with rubber septa and purged with N2 gas for 30 minutes. The solutions were then placed on an oil bath set atop a hot plate. The hot plate was set to 60oC and 220 rpm stirring for 3.5 hours. Following taking the copolymers off the oil bath, to ensure that polymerization had occurred, samples were taken for both 1H NMR and GPC analysis for verification. 1H NMR for the copolymers utilized for nanodisc formation are presented in the supplemental figures section, in Figures S1 and S2.

Precipitation of VEMA Copolymers

In order to purify the copolymer from any remaining unreacted monomers, CTA, initiator, or dioxane, the VEMA copolymers were precipitated in cold hexanes. To do so, two 100mL beakers were filled with approximately 60mL of hexanes and placed on an ice bucket for 10 minutes. Each of the copolymers were dissolved in a minimal amount of tetrahydrofuran (THF), added until a viscosity similar to water was achieved. The polymers were then each added dropwise into respective beakers. If the copolymer appeared gel-like and not solid, as much cold hexanes were decanted as possible, and then precipitation in the same manner was completed again. When the copolymer appeared solid after cold hexanes were decanted, the beaker was then covered with a layer of parafilm with holes to allow for volatile liquid to escape, and placed in a vacuum dessicator for 24 hours, until appearing powder-like and dry.

Hydrolysis of VEMA Copolymers

A portion of the copolymers obtained (approximately 0.5000g) was utilized for hydrolysis, allowing for study of vesicles formed with neither Alanine-D or Alanine-L functionalization. Hydrolysis was performed by mixing 2.75mL of 4M sodium hydroxide (NAOH) with 12mL of water, and adding this solution drop-wise to a 50mL round-bottom flask containing 0.5g of polymer dissolved in minimal THF on an oil bath, stirring at 220rpm and heated at 55oC. A rubber septum used as a stopper for the round-bottom flask was utilized to prevent any accidental contamination of the polymer during the hydrolysis reaction, and the reaction was left for 24 hours prior to removal from the oil bath.

Alanine Functionalization of VEMA Copolymers

For each copolymer A and B, functionalization with Alanine-D and Alanine-L were performed, following a previously outlined protocol for similar copolymers that instead used styrene rather than vinyl ether monomers4. To functionalize with alanine, approximately 300mg of copolymer was added to a 20mL glass vial. Each vial, with copolymers being approximately 3mmol/g anhydride, thus had 0.9 mmol anhydride. The copolymer was then dissolved in minimal dimethylformamide (DMF). Simultaneously to the vial were added triethylamine (TEA) (172.3uL, 1.24mmol) and either H-Ala-OH (L-Alanine) or H-D-Ala-OH (D-Alanine) (.1101g, 1.24mmol). The solution was then stirred at room temperature in the 20mL glass vial at 220rpm until a solution was achieved. The copolymer was then precipitated by adding the solution drop-wise to a 100mL beaker with approximately 40mL of rapidly stirring diethyl ether, which had been chilled on an ice bath for 10 minutes. The diethyl ether was then decanted, and the copolymer was then washed twice with 20mL of ethyl acetate chilled on an ice bath for 10 minutes. The ethyl acetate was mixed well with the copolymer prior to decanting. The copolymer was then dried in a vacuum dessicator for a minimum of 30 minutes at a low temperature of approximately 50oC to remove any volatiles.

The vacuum-dried samples were then suspended by mixing well in approximately 10mL of 0.1M HCl in a 15mL centrifuge tube. The tube was then spun at 6k rpm for 6 minutes. The HCl was decanted, and resuspension was completed again with an additional 10mL of 0.1M HCl. The tubes were spun again at 6k rpm for 6 minutes, and the HCl was decanted. If a pellet was not achieved, the tubes were again resuspended with approximately 10mL of 0.1M HCl and spun at 6k rpm for up to 15 minutes, where HCl was then decanted. The polymer was then freeze-dried, yielding approximately 80mg of off-white powder. Conversion of the anhydride was then confirmed by infrared spectroscopy as well as 1H NMR.

POPC Vesicle Preparation

A previously published procedure3 to prepare the 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles from the D-Alanine and L-Alanine functionalized copolymers was utilized. POPC was utilized over other synthetic liposomes as the phosphatidylcholine head present in the vesicles is the most highly abundant head group that is found in eukaryotic cell membranes. The POPC powdered lipid was dissolved in a buffer which contained 100mM NaCl and 20mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) at pH 7.0, with a POPC concentration of 25mM. The lipid slurry that was then formed was vortexed to mix completely, which allowed for spontaneous formation of lipids. 10 cycles of freeze/sonication at less than 30oC allowed for the formation of a homogeneous, milky solution, which was then frozen with liquid nitrogen, and the solution was placed in a freezer overnight at -20oC. To confirm the vesicle size, dynamic light scattering (DLS) was then performed. 

Transmission electron microscopy images were then recorded by the placement and absorbance of a drop of sample onto 200mesh copper carbon-coated grids, with 10 seconds of contact to ensure full absorbance. The copper carbon-coated grids were then stained with two drops of 1.5% ammonium molybdate and images were obtained.

Despite functionalization of the polymers with D- and L-alanine having identical effects as seen from the results of dynamic light scattering (DLS), functionalization of vinyl ether maleic anhydride polymers with other, larger amino acids may allow for differences in rigidity and dynamics to be seen, which are dependent on the amino acid functional groups. Performing circular dichroism (CD) and pH analysis of chiral amino acid polymers may also potentially elucidate differences existing between charged enantiomeric polymers. Utilizing additional vinyl ethers, such as cyclohexyl vinyl ether, may also prove to show differences in DLS or other experiments based on amino acid functionalization.

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This project was completed by researchers who have completed the Collaborative Institution Training Initiative (CITI) for a basic introduction to biosafety, NIH recombinant DNA guidelines, and OSHA bloodborne pathogens, and executed under a Miami University approved Chemical Hygiene Plan.