PEG length and chemical linkage controls polyacridine peptide DNA polyplex pharmacokinetics, biodistribution, metabolic stability and in vivo gene expression

Abstract

The pharmacokinetics (PK), biodistribution and metabolism of non-viral gene delivery systems administered systemically are directly related to in vivo efficacy. The magnitude of luciferase expression in the liver of mice following a tail vein dose of a polyplex, composed of 1 μg of pGL3 in complex with a polyethylene glycol (PEG) polyacridine peptide, followed by a delayed hydrodynamic (HD) stimulation (1–9 h), depends on the HD stimulation delay time and the structure of the polyacridine peptide. As demonstrated in the present study, the PEG length and the type of chemical linkage joining PEG to the polyacridine peptide dramatically influence the in vivo gene transfer efficiency. To understand how PEG length, linkage and location influence gene transfer efficiency, detailed PK, biodistribution and HD-stimulated gene expression experiments were performed on polyplexes prepared with an optimized polyacridine peptide modified through a single terminal Cys or Pen (penicillamine) with a PEG chain of average length of 2, 5, 10, 20, or 30 kDa. The chemical linkage was examined by attaching PEG5kDa to the polyacridine peptide through a thiol-thiol (SS), thiol-maleimide (SM), thiol-vinylsulfone (SV), thiol-acetamide (SA), penicillamine-thiol-maleimide (PM) or penicillamine-thiol-thiol (PS). The influence of PEG location was analyzed by attaching PEG5kDa to the polyacridine peptide through a C-terminal, N-terminal, or a middle Cys residue. The results established rapid metabolism of polyplexes containing SV and SA chemical linkages leads to a decreased polyplex PK half-life and a complete loss of HD-stimulated gene expression at delay times of 5 hrs. Conversely, polyplexes containing PM, PS, and SM chemical linkages were metabolically stable, allowing robust HD-stimulated expression at delay times up to 5 hrs post polyplex administration. The location of PEG5kDa within the polyacridine peptide exerted only a minor influence on the gene transfer of polyplexes. However, varying the PEG length from 2, 5, 10, 20, or 30 kDa dramatically altered polyplex biodistribution, with a 30 kDa PEG maximally blocking liver uptake to 13% of dose, while maintaining the ability to mediate HD-stimulated gene expression. The combination of results establishes important relationships between PEGylated polyacridine peptide structure, physical properties, in vivo metabolism, PK and biodistribution resulting in an optimal PEG length and linkage that leads to robust HD-stimulated gene expression in mice.

“Evolving nanoparticle gene delivery vectors for the liver: What has been learned in 30 years”

“…By modifying the hydrodynamic delivery procedure the method can also be used to study the circulatory stability of nonviral gene delivery vectors under development [57][58][59]. Delayed hydrodynamic stimulation is similar to direct hydrodynamic delivery, except the DNA and saline are separated into different injections.…”

“…If the circulating DNA remains intact, gene expression in the liver is equivalent to that achieved by direct hydrodynamic delivery. Delayed hydrodynamic stimulation has been applied to study the circulatory stability of DNA nanoparticles [60][61][62][63]. Biodistribution studies established stable DNA nanoparticles cross the liver fenestrae and transiently move through the space of Disse, prior to hydrodynamic stimulation into hepatocytes [64].…”

“…dosing these circulated in mice for several hours. Stable DNA polyplexes remained fully transfection competent in the circulation for up to four hours as demonstrated by administration of hydrodynamic stimulation which resulted in gene expression in the liver [62,63].…”

“…One set of receptors used to carry out this function is collectively known as "scavenger receptors", which capture anionic particles [122][123][124][125][126]. Scavenger receptors are responsible for first-pass capture of approximately 60% of the PEGylated DNA nanoparticles by the liver [63,[127][128][129][130][131][132][133][134][135]. Viruses, liposomes, naked plasmid DNA, and PEGylated DNA peptide polyplexes are taken up by the liver through two mechanisms [64,118,136,137], a rapid uptake and a delayed uptake.…”

Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

“…1,4 A safe and efficient delivery system is thus needed for a successful gene therapy. 1,4,5 Since 1960s, nonviral vectors based on cationic polymers [6][7][8][9] and lipids…”