Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry

Abstract

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein "scaffold," which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo-lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine "branches" are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell-free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo-EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6-30 nm) and polydispersity. The telodendrimer NLPs (TD-NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.

Cryo‐electron microscopy and cryo‐electron tomography of nanoparticles

“…Many cryo‐TEM studies have been performed on lipid based nanoparticles, including liposome, lipopeptide, lipid liquid crystalline, and solid lipid nanoparticles . Lyotropic liquid crystalline nanoparticles represent a class of lipid‐based particles, which have highly ordered, internal water channels and which have strong potential for drug delivery applications .…”

Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles

“…We have previously detailed the use of telodendrimers as a nano‐delivery tool . PEG5k‐CA8 was prepared according to published methods .…”

“…To that end, we performed simultaneous Small‐Angle Neutron Scattering and Small‐Angle X‐ray Scattering (SANS/SAXS) on NLPs assembled from 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) lipids produced via traditional in vitro methods, and by the cell‐free method. We also analyzed NLPs produced using the cell‐free method in the presence of a PEG5k‐CA8 telodendrimer additive, which has been shown to improve solution stability and monodispersity of the NLPs . Finally, after verifying the structural equivalence of the three NLP preparations, we fit an appropriate core‐shell disc‐type model to the scattering data to verify the geometry and molecular composition of NLPs.…”

“…We also analyzed NLPs produced using the cell-free method in the presence of a PEG5k-CA8 telodendrimer additive, which has been shown to improve solution stability and monodispersity of the NLPs. 5,6 Finally, after verifying the structural equivalence of the three NLP preparations, we fit an appropriate core-shell disc-type model to the scattering data to verify the geometry and molecular composition of NLPs. To improve the robustness of the model fitting, we also included SANS data from an NLP sample prepared using tail-deuterated DMPC.…”

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

“…liposomes) for vaccine delivery. The procedure of NLP synthesis is very flexible as lipid and protein components can be easily substituted to accommodate various membrane proteins or achieve desired chemical properties (22)(23)(24). NLPs also enhance membrane protein solubility and in vivo delivery (25).…”