Metabolically stabilized long-circulating PEGylated polyacridine peptide polyplexes mediate hydrodynamically stimulated gene expression in liver

Abstract

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.

Folic‐Acid‐Mediated Functionalized Gold Nanocages for Targeted Delivery of Anti‐miR‐181b in Combination of Gene Therapy and Photothermal Therapy against Hepatocellular Carcinoma

“…This result was due to the prolonged circulation time induced by PEG conjugated on the surface of AuNC-NPs, as previously reported. [39][40][41] These observations indicate that the FA-mediated targeted PTPAuNC-NPs facilitated the accumulation of gene drugs in tumors, compared with naked anti-miR-NC and passive targeted formulation, PPAuNC-NPs.…”

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

“…Its use has made it possible to examine gene regulation [53], determine the effects of transgene expression on animal disease models [54], and establish the efficacy of siRNA knockdown in liver [40,42,55]. In addition, it has become the benchmark method for gene transfer efficiency to which all other systemic nonviral delivery methods to transfect the liver have been compared [56,57].…”

“…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.…”

“…To increase the affinity of short peptides for DNA, several groups have incorporated multiple intercalators into vectors to hydrophobically bind to DNA [97][98][99] The intercalator acridine, can be most easily added to the side chain of lysine, then installed into a peptide at any desired location during solid phase peptide synthesis [61,100]. Polyacridine peptides ( Fig.…”

“…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].…”