Endosomolytic Reducible Polymeric Electrolytes for Cytosolic Protein Delivery

Abstract

Despite the numerous vital functions of proteins in the cytosolic compartment, less attention has been paid to the delivery of protein drugs to the cytosol than to the plasma membrane. To address this issue and effectively deliver charged proteins into the cytoplasm, we used endosomolytic, thiol-triggered degradable polyelectrolytes as carriers. The cationic, reducible polyelectrolyte RPC-bPEI0.8kDa2 was synthesized by the oxidative polymerization of thiolated branched polyethyleneimine (bPEI). The polymer was converted to the anionic, reducible polyelectrolyte RPA-bPEI0.8kDa2 by introducing carboxylic acids. The two reducible polyelectrolytes (RPC-bPEI0.8kDa2 and RPA-bPEI0.8kDa2) were complexed with counter-charged model proteins (bovine serum albumin (BSA) and lysozyme (LYZ)), forming polyelectrolyte/protein complexes of less than 200 nm in size at weight ratios (WR) of ≥ 1. The resultant complexes maintained a proton buffering capacity nearly equivalent to that of the polyelectrolytes in the absence of protein complexation and were cytocompatible with MCF7 human breast carcinoma cells. Under cytosol-mimicking thiol-rich conditions, RPC-bPEI0.8kDa2/BSA and RPA-bPEI0.8kDa2/LYZ complexes increased significantly in size and released the loaded protein, unlike the protein complexes with non-reducible polyelectrolytes (bPEI25kDa and bPEI25kDaCOOH). The polyelectrolyte/protein complexes showed similar cellular uptake to the corresponding proteins alone, but the former allowed more protein to escape into the cytosol from endolysosomes than the latter as a result of the endosomolytic function of the polyelectrolytes. In addition, the proteins in the polyelectrolyte/protein complexes kept their intrinsic secondary structures. In conclusion, the results show the potential of the designed endosomolytic, reducible polyelectrolytes for the delivery of proteins to the cytosol.

Oncolytic Adenovirus Coated with Multidegradable Bioreducible Core-Cross-Linked Polyethylenimine for Cancer Gene Therapy

“…A previous report showed that the cationic polymers induce cytotoxicity by interacting with intracellular components and that higher molecular weight polymers have stronger interaction with these components, which results in higher toxicity. 42 The viability of the cells treated with either rPEI (16 kDa, 32 kDa) was above 76.8%, Characterization of Ad/Polymer Complex. The formation of Ad/polymers nanoparticles was driven by electrostatic interaction between Ad and rPEIs.…”

Bioreducible Branched Polyethyleneimine Derivatives Physically Loaded with Hydrophobic Pheophorbide A: Preparation, Characterization, and Light‐Induced Cytotoxicity

“…First, if acidic pH values can induce PheoA release from bPEI 25kDa ‐PheoA, then bPEI 25kDa ‐PheoA would need to be exposed to intracellular acidic environments such as those of late endosomes or lysosomes. However, the endosomal escape ability of bPEI 25kDa could prohibit the exposure of bPEI 25kDa ‐PheoA to these acidic compartments. Second, if bPEI 25kDa ‐PheoA enters the cell through direct membrane penetration (given that polycations such as bPEI can penetrate through the plasma membrane and detour from endocytic pathways), bPEI 25kDa ‐PheoA would be located in the cytosol after cellular internalization.…”

Bioreducible Polycations in Nucleic Acid Delivery: Past, Present, and Future Trends

“…Various methods of thiolation have been reported (Figure ). Among the most common are methods that use reaction of iminothiolane with primary amines of a polycation such as PEI and poly( L ‐lysine) (PLL) . Using iminothiolane conserves the number of positive charges of the polycation and is applicable to both conventional synthesis of polycations as well as in situ formation of bioreducible polycations after polyplex formation with thiolated polycations.…”