PEGylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that (Acr-Lys)6-Cys-PEG5kDa stabilizes a 1 μg pGL3 dose for up to 1 hr in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 μg of pGL3 (Fernandez C.A. et al. Gene Therapy 2011). In the present study we report that increasing the spacing of Acr with either 4 or 5 Lys residues, dramatically increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 hrs post-DNA administration. Co-administration of a decoy dose of 9 μg of non-expressing DNA polyplex with 1 μg of pGL3 polyplex further extended the HD-stimulated expression to 9 hrs. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 hrs and provides an understanding as to why polyplexes or lipoplexes prepared with PEI, chitosan or Lipofectamine are inactive within 5 min following i.v. dosing.

Two uptake mechanisms were identified for PEGylated DNA polyplex biodistribution to the liver. At a low polyplex dose, a rapid-uptake mechanism dominates, resulting in 60% capture by liver in 5 min, due to a saturable receptor-mediated process. Rapid-uptake led to the fast metabolism of polyplexes by liver (t1/2 = 2.1 h), correlating with a 1-μg pGL3 polyplex dose losing full transfection competency after 4 h in the liver. Dose escalation of either polyplex or poly(ethylene glycol) (PEG) peptide led to the saturation of rapid-uptake and revealed a delayed-uptake mechanism for polyplexes by liver. Delayed-uptake was characterized by the slower liver accumulation of 40% of the polyplex dose over 40 min, followed by slow metabolism (t1/2 = 15 h) and an extended time (12 h) for a 1-μg pGL3 polyplex dose, remaining fully transfection competent in the liver. The delayed-uptake mechanism is consistent with polyplexes crossing liver fenestrated endothelial cells to reach steady state in the space of Disse. The results describe how to control polyplex biodistribution to liver to avoid rapid-uptake and metabolism, in favor of delayed-uptake, to preserve polyplex transfection competency in the liver for up to 12 h.

A novel class of PEGylated polyacridine peptides was developed that mediate potent stimulated gene transfer in the liver of mice. Polyacridine peptides, (Acr-X)n-Cys-PEG, possessing 2–6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with polyethylene glycol (PEG 5000 Da) and evaluated as in vivo gene transfer agents. An optimal peptide of (Acr-Lys)6-Cys-PEG was able to bind to plasmid DNA (pGL3) with high affinity by polyintercalation, stabilize DNA from metabolism by DNAse and extend the pharmacokinetic half-life of DNA in the circulation for up to 2 hrs. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 μg in 50 μl), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5–60 min post-DNA administration, led to a high level of luciferase expression in the liver, equivalent to levels mediated by direct hydrodynamic dosing of 1 μg of pGL3. The results establish the unique properties of PEGylated polyacridine peptides as a new and promising class of gene delivery peptides that facilitate reversible binding to plasmid DNA, protecting it from DNase in vivo resulting in an extended circulatory half-life, and release of transfection-competent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost.

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.

PEGylated polylysine peptides of the general structure PEG30kDa-Cys-Trp-Lys (N =10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per μg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of 125I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased ten-fold when comparing PEG30kDa-Cys-Trp-Lys10 (IC50 of 20.2 μM) with PEG30kDa-Cys-Trp-Lys25 (IC50 of 2.1 μM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.