Cationic α‑Cyclodextrin:Poly(ethylene glycol) Polyrotaxanes for siRNA Delivery

RNA interference has broad therapeutic potential due to its high specificity and ability to potentially evade drug resistance. Three cationic α-cyclodextrin:poly­(ethylene glycol) polyrotaxanes derived from polymer axles of different sizes (MW 2,000, 3,400, and 10,000) have been synthesized for delivering siRNA. These polyrotaxanes are able to condense siRNA into positively charged particles that are <200 nm in diameter, enabling their facile internalization into mammalian cells. The cationic polyrotaxanes display cytotoxicity profiles that are >10²-fold lower than the commercial standard bPEI and gene silencing efficiencies that are comparable to those of both Lipofectamine 2000 and bPEI. Our findings suggest that the cationic polyrotaxanes display a size–activity relationship, wherein the higher molecular weight polyrotaxanes (PEG3,400 and 10,000) are able to condense and deliver siRNA better than the lower molecular weight material (PEG2,000)

Organocatalytic Synthesis and Evaluation of Polycarbonate Pendant Polymer:β-Cyclodextrin-Based Nucleic Acid Delivery Vectors

A family of mPEG-<i>b</i>-polycarbonate (mPEG-PC) diblock pendant polymers were synthesized from trimethylene carbonate and other cyclic carbonate monomers bearing hydrophobic guest ligands via organocatalytic ring-opening polymerization using 1,4,5-triazabicyclo[4.4.0]­dec-5-ene catalyst or 1,8-diazabicyclo[5.4.0]­undec-7-ene/thiourea cocatalyst. Diblock copolymers composed of a methoxy­poly­(ethylene oxide) (mPEG) block and a polycarbonate block containing either homopolymer or mixed polycarbonates (PC) were prepared by homopolymerization or copolymerization of the cyclic carbonate monomers in the presence of mPEG2000 or mPEG5000 initiator to give materials having a tunable pendant group density along the polycarbonate backbone. Polycarbonate blocks targeting the 2.4–10 kDa range were prepared with good molecular weight control and modest polydispersities (averaging ∼1.3). Complexation of plasmid DNA with β-cyclodextrin–polyethylenimine2.5 kDa produced nanoparticle cores that were then coated with the mPEG–PC diblock copolymers to produce transfection complexes in the 100–250 nm size range. Stable transfection complexes prepared at N/P ratios >10 had slightly positive ζ potentials and showed comparable or modestly better transfection efficiencies in HeLa cells than the commercial transfection agent, Lipofectamine2000. Transfection efficiencies were not dependent on polycarbonate block molecular weights. The mPEG-PC constructs displayed similar efficacy for adamantyl and cholesteryl pendants that strongly bind to β-cyclodextrin; however, slightly better performance was observed for the weakly bound pendant, benzyl. These findings suggest that pDNA release is largely mediated by hydrolysis of the ester-bound pendant ligand within the endolysosomal compartment of the cell, with desorption of the mPEG–PC layer also contributing to plasmid release and activation in the case of weak binding pendant groups. We infer from these results that mPEG-PC may be an effective degradable transfection agent for <i>in vivo</i> applications

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates

We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly­(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir–Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine-<i>N</i>-[(5-amido-1-carboxypentyl)­iminodiacetic acid]­polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10′,12′-diynoyl)-<i>sn</i>-glycero-3-phosphoethanolamine-<i>N</i>-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His₆-T7 bacteriophage and RplL from cell lysates, as well as purified His₈-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His₆-MalFGK₂. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure