Intracellular Delivery of Active Proteins by Polyphosphazene Polymers

Abstract

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

Nano-Assembly of Quisinostat and Biodegradable Macromolecular Carrier Results in Supramolecular Complexes with Slow-Release Capabilities

“…The results of these studies can be reviewed considering two outermost mechanistic modalities: potential activity of the entire multimeric complex as a single entity (dose-effect relationship analyzed vs. concentration of the complex-Figures 4A and 5B) and, more traditionally, assuming that quisinostat is active once its molecules are detached from the polymer, analysis of dose-effect curves plotted against concentration of a drug (Figures 4B and 5B). The first hypothesis may be considered taking into account established ability of PEGylated polyphosphazenes to facilitate uptake and intracellular delivery of its cargo, which has been demonstrated mainly for delivery of proteins and peptides [22,49,50]. The second assumption can be supported by the ongoing release of quisinostat under the conditions similar to those of viability experiments (Figure 3A,B).…”

“…We recently introduced anionic polyphosphazenes with biodegradable backbone containing graft poly(ethylene glycol) PEG chains (PPEGs), which were characterized by improved water-solubility and stability to aggregation [21]. These polymers were explored as noncovalent PEGylation agents for extending half-life of proteins [21] and intracellular delivery of peptide and protein cargo, and were shown to be effective in facilitating cellular uptake of protein cargo and non-toxic to cells [22]. Applications of such PEGylated macromolecules to the delivery of HDACis can offer a simple single-step formulation alternative to more sophisticated and labor intense methodologies.…”

Polyphosphazenes—A Promising Candidate for Drug Delivery, Bioimaging, and Tissue Engineering: A Review

“…Ionic PPZs can combine noncovalently with various kinds of protein cargo to form supramolecular complexes. [ 120 ] PEGylated graft copolymer (PPZ‐PEG) (PPZ R30) and a pyrrolidone containing linear derivative (PPZ‐PYR) (PPZ R31) can effectively carry huge amounts of large and tiny protein cargos by noncovalent complexation. [ 120 ] Proteins frequently suffer from short in vivo half‐lives.…”

“…[ 120 ] PEGylated graft copolymer (PPZ‐PEG) (PPZ R30) and a pyrrolidone containing linear derivative (PPZ‐PYR) (PPZ R31) can effectively carry huge amounts of large and tiny protein cargos by noncovalent complexation. [ 120 ] Proteins frequently suffer from short in vivo half‐lives. [ 121 ] Hence injectable controlled release formulations are desperately needed.…”

Comparison between Nanoparticle Encapsulation and Surface Loading for Lysosomal Enzyme Replacement Therapy

“…Among these listed nanocarriers, polymer-based NPs hold great potential for protein delivery due to their stability and multifunctionality [ 22 ]. Polymeric NPs can be prepared using either biodegradable synthetic polymers such as poly(lactide-co-glycolide) (PLGA) copolymers, polyacrylates, poly(caprolactone)s, polyphosphazenes, or natural polymers such as albumin, gelatin, alginate, collagen, or chitosan [ 14 , 22 , 23 , 24 , 25 ].…”