Lipodisq™ Styrene:Maleic Acid Universal Copolymer with pH-Selector

• A nanoparticle (11-40nm) drug delivery system comprising a discoidal phospholipid bilayer membrane stabilized by a chaperone molecular annulus.
• Components are batch-tested for Lipodisq™ formation using buffer systems available, which are tested for nano-formulated drug analysis by Dynamic Light Scattering (DLS). These buffer solutions are endotoxin-tested and sterile.
• Lipodisq™ formation is highly efficient and shows a good safety profile, suitable for in vitro and in vivo (in experimental animals) investigations.

Enables membrane proteins (MP) to be reconstituted within phospholipid membrane mimics without the use of mediating detergent. Nanodiscs formed from SMA are also referred to as styrene maleic acid lipid particles (SMALP). SMA-lipid particles are formed by directly extracting membrane proteins either from native cellular membranes (giving native nanodiscs) or from an intermediary MP-reconstituted synthetic membrane system to ultimately form self-assembled discoidal nano-structures of a general diameter of 10-12 nm.

• uSMA requires only a single polymer grade to be used, avoids the need to select several SMA grades that each operate only over a narrow pH range.
• uSMA can be tailored to operate over a wide pH range 4.5-8.5 by simple addition of standard quantities of pH-Selector™
• uSMA is insensitive to presence of divalent cations, Ca2+(<10mM) and Mg2+ (<30mM), suitable for use with ion pores, ABC transporters and ion sensitive GPCR assemblies
• uSMA can be modified to form larger particles, reducing direct interactions between the polymer chaperone and membrane‑bound proteins

The ability of SMA to create monodisperse nanodiscs covering a range of particle sizes facilitates the reconstitution of oligomeric MPs and MP complexes for analysis by various methods including fluorescence microscopy, NMR and single-particle cryo-EM.

• As a starting point use a ratio of 2.5:1 (w/w) for P(SMA)/SMA polymer and phospholipid, such as 100mg P(SMA)/SMA polymer and 40mg DMPC. Prepare 5% (w/w) uSMA in deionized water.  
• Adjust the solution with the corresponding volume of pH-Selector (see table) and confirm the pH.
• Prepare a 2% (w/w) aqueous phospholipid emulsion in deionized water. Incorporate hydrophobic drug candidate/peptides/transmembrane proteins (MP) at up to 0.2% (w/w)
• This mixture is stirred at temperatures above the phase transition temperature of the lipid  (>24°C for DMPC), before aqueous P(SMA)/SMA polymer at 5% is added drop-wise until  an approximate volume ratio of 1:1 is reached and the lipid emulsion clears.
• Alternatively, P(SMA)/SMA polymer solutions containing the pH selectorTM at the selected pH are mixed with native (cell or bacterial) membranes to form native nanodiscs.
• Stirring time, pH, selected buffer type and strength (e.g. HEPES, NaCl, TRIS or PBS with or w/o Ca++ and Mg++), pH-SelectorTM and temperature of the phospholipid emulsion containing the MP or active agent, need to be optimized.
• Purification of formed LipodisqTM can be achieved by ultracentrifugation at >100,000 x g to remove residual lipid and excess P(SMA)polymer or a size exclusion column (SEC) can be used.