Hydrolytically Degradable PEGylated Polyelectrolyte Nanocomplexes for Protein Delivery

Abstract

Novel oppositely charged polyphosphazene polyelectrolytes containing grafted poly(ethylene glycol) (PEG) chains were synthesized as modular components for the assembly of biodegradable PEGylated protein delivery vehicles. These macromolecular counterparts, which contained either carboxylic acid or tertiary amino groups, were then formulated at near physiological conditions into supramolecular assemblies of nanoscale level, below 100 nm. Nanocomplexes with electroneutral surface charge, as assessed by zeta potential measurements, were stable in aqueous solutions, which suggests their compact polyelectrolyte complex "core"-hydrophilic PEG "shell" structure. Investigation of PEGylated polyphosphazene nanocomplexes as agents for noncovalent PEGylation of the therapeutic protein l-asparaginase (L-ASP) in vitro demonstrated their ability to dramatically reduce protein antigenicity, as measured by antibody binding using enzyme linked immunosorbent assay (ELISA). Encapsulation in nanocomplexes did not affect enzymatic activity of L-ASP, but improved its thermal stability and proteolytic resistance. Gel permeation chromatography (GPC) experiments revealed that all synthesized polyphosphazenes exhibited composition controlled hydrolytic degradability in aqueous solutions at neutral pH and showed greater stability at lower temperatures. Overall, novel hydrolytically degradable polyphosphazene polyelectrolytes capable of spontaneous self-assembly into PEGylated nanoparticulates in aqueous solutions can potentially enable a simple and effective approach to modifying therapeutic proteins without the need for their covalent modification.

Polyphosphazenes for the delivery of biopharmaceuticals

“…This material showed low toxicity and good capacity to support cell adhesion, proliferation, and maintenance of cellular phenotype . Other types of PEGylated PPZs also show excellent biosafety profiles that combine with their antifouling properties . The biocompatibility of the PPZ backbone has also been supported by the FDA‐approval of Cobra PzF stents (CeloNova Biosciences Inc., US) in 2017, which are coronary stents coated with nanostructured PPZs for thromboresistant properties …”

“…Carboxylatoethylphenoxy (CEP) side chains are well known for their use in complexation with proteins, but they are also sensitive to changes in pH within the physiological range. Complexation of L‐Asparaginase with both negatively charged CEP‐grafted and positively charged tertiary amine‐grafted PEG‐PPZ derivatives can form noncovalent PEGylated nanoassemblies for protein delivery; these nanocarriers promoted the stability of this enzyme and reduced their undesirable antigenicity …”

Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us

“…Complex coacervation, [49,51,52,56,57,[61][62][63][64][65][66][67][68][69][70][71][72][73] whose characteristics mimic the characteristics of cells, is a simple, purely aqueous strategy for encapsulating proteins. Complex coacervation is a liquid-liquid phase separation phenomenon resulting from the association of two oppositely charged macro-ions (e.g., polymer, protein, surfactant micelles).…”

“…While the results to date have focused on complex coacervates that undergo macrophase separation, it is also possible to take advantage of block copolymer architectures to facilitate hierarchical microphase separation. In particular, a majority of efforts have coupled a water soluble polymer block, such as poly(ethylene glycol) (PEG) [69,110] or poly(oligoethylene glycol methacrylate) (POEGMA) [91] to one or both of the oppositely-charged polymers. This block copolymer structure creates an interface that drives microphase separation, with the relative length of the two blocks dictating the resulting structure.…”

Intracellular Delivery of Active Proteins by Polyphosphazene Polymers

“…Water-soluble polyphosphazenes (PZs) are hybrid organic-inorganic polyelectrolyte-type polymers that are attaining increasing recognition as multifunctional drug delivery vehicles [27][28][29]. Their unique macromolecular structure consists of an alternating phosphorus and nitrogen backbone and organic side groups, which provides for hydrolytic degradability under physiological conditions [30] and enables self-assembly with macromolecular drugs, including proteins, and some biological targets through non-covalent interactions [27,[31][32][33][34]. PZ polymers have been formulated mostly as vaccine adjuvants [35] or in supramolecular assemblies including micelles, nanoparticles, hydrogels etc., hence they are flexible systems with multiple applications accorded by adjusting the side groups for different applications [36].…”