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

• Published in: Journal of controlled release Vol. 170; no. 3; pp. 325 - 333
• Main Authors: Khargharia, Sanjib, Kizzire, Koby, Ericson, Mark D., Baumhover, Nicholas J., Rice, Kevin G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 28.09.2013
• Subjects: Acridines - chemistry; Animals; blood veins; DNA; DNA - administration & dosage; DNA - chemistry; DNA - pharmacokinetics; Gene delivery; Gene Expression; gene transfer; Gene Transfer Techniques; genes; half life; Hydrodynamic dosing; hydrodynamics; liver; luciferase; Luciferases - genetics; Male; Mice; Mice, Inbred ICR; penicillamine; Peptides - administration & dosage; Peptides - chemistry; Peptides - pharmacokinetics; pharmacokinetics; physical properties; Polyethylene glycol; Polyethylene Glycols - chemistry; Polymers - chemistry; tail; Tissue Distribution; Gene delivery; Polyethylene glycol; Hydrodynamic dosing
• ISSN: 0168-3659; 1873-4995
• DOI: 10.1016/j.jconrel.2013.05.024

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 1μg of pGL3 in complex with a polyethylene glycol (PEG) polyacridine peptide, followed by a delayed hydrodynamic (HD) stimulation (1–9h), 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 30kDa. 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 that leads to a decreased polyplex PK half-life and a complete loss of HD-stimulated gene expression at delay times of 5h. Conversely, polyplexes containing PM, PS, and SM chemical linkages were metabolically stable, allowing robust HD-stimulated expression at delay times up to 5h 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 30kDa dramatically altered polyplex biodistribution, with a 30kDa 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 a robust HD-stimulated gene expression in mice. [Display omitted]