The Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery (original) (raw)
Related papers
PEG-Peptide Inhibition of Scavenger Receptor Uptake of Nanoparticles by the Liver
Molecular Pharmaceutics, 2018
PEGylated polylysine peptides represent a new class of scavenger receptor inhibitors that may find utility at inhibiting DNA nanoparticle uptake by Kupffer cells in the liver. PEG-peptides inhibit scavenger receptors in the liver by a novel mechanism involving in situ formation of albumin nanoparticles. The present study developed a new in vivo assay used to explore the structureactivity-relationships of PEG-peptides to find potent scavenger receptor inhibitors. Radioiodinated PEG-peptides were dosed i.v. in mice and shown to saturate liver uptake in a dosedependent fashion. The inhibition potency (IC 50) was dependent on both the length of a polylysine repeat and PEG molecular weight. PEG 30kda-Cys-Tyr-Lys 25 was confirmed to be a high molecular weight (33.5 kDa) scavenger receptor inhibitor with an IC 50 of 18 μM. Incorporation of multiple Leu residues compensated, to allow a decrease in PEG MW and Lys repeat, resulting in PEG 5kda-Cys-Tyr-Lys-(Leu-Lys 4) 3-Leu-Lys that inhibited scavenger receptors with an IC 50 = 20 μM. A further decrease in PEG MW to 2 kDa increased potency, resulting in a low molecular weight (4403 g/mol) PEG-peptide with an IC 50 of 3 μM. Optimized low molecular weight PEG-peptides also demonstrated potency when inhibiting the uptake of radio-iodinated DNA nanoparticles by the liver. This study demonstrates an approach to discover low molecular weight PEG-peptides that serve as potent scavenger receptor inhibitors to block nanoparticle uptake by the liver.
Synthetic PEGylated Glycoproteins and Their Utility in Gene Delivery
Bioconjugate Chemistry, 2007
PEGylated glycoproteins (PGPs) were synthesized by co-polymerizing a Cys-terminated PEGpeptide, glycopeptide and melittin peptide. Compositionally unique PGPs were prepared by varying the ratio of PEG-peptide (20%-90%) and melittin (0-70%) with a constant amount of glycopeptide (10%). The PGPs were purified by RP-HPLC, characterized for molecular weight and polydispersity by GPC-HPLC and SDS-PAGE, and for composition by RP-HPLC following reduction to form monomeric peptides. PGPs formed DNA condensates of 200-300 nm in diameter that were administered to mice via the tail vein. Biodistribution studies confirmed their primary targeting to liver hepatocytes with a DNA metabolic half-life of 1 hour. Upon stimulation by hydrodynamic dosing with saline, PGP DNA (5 μg) mediated luciferase expression in the liver detected by bioluminescence imaging (BLI) after 24 hours. The level of gene expression mediated by PGP DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP. The results establish the ability to prepare three-component gene delivery polymers that function in vivo. Further design improvements in fusogenic peptides for gene delivery and for the simultaneous use of a nuclear targeting strategy will be necessary to approach levels of expression mediated by the direct hydrodynamic dosing of DNA.
European Journal of Cell Biology, 2004
In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear bcyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.
Gene Therapy, 2011
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-polyethylene glycol (PEG), possessing 2-6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with PEG (PEG 5000 kDa ) 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 h. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 mg in 50 ml), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5 to 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 mg 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 transfectioncompetent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost.
Journal of Controlled Release, 2013
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 PEG 5kDa 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 PEG 5kDa to the polyacridine peptide through a C-terminal, Nterminal, 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 HDstimulated expression at delay times up to 5 hrs post polyplex administration. The location of PEG 5kDa 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.
A novel PEGylation of chitosan nanoparticles for gene delivery
Biotechnology and Applied Biochemistry, 2007
CS (chitosan) has emerged as a promising non-viral vector for gene delivery because of its ability to form complexes with pDNA (plasmid DNA) and enhance its transport across cellular membranes through endocytosis. Complexes of CS and pDNA may improve transfection efficiency; however, they are not capable of sustained DNA release and prolonging gene transfer. In order to achieve prolonged delivery of CS-DNA complexes, we prepared CS NP (nanoparticle) and CS-DNA complexes. α-Methoxy-ω-succinimidylpoly(ethylene glycol) was then conjugated to the surface of CS-DNA complexes using an active ester scheme; finally, the potential of PEGylation [poly(ethylene glycol)ylation] of CS NP as a non-viral gene-delivery vector to transfer exogenous genes in vitro and in vivo were examined. Electrophoretic analysis suggested that CS NPs could protect the DNA from nuclease degradation. The pDNA carried by CS NPs could enter and be expressed in HepG2 cells. However, the transfection efficiency was very low and the highest dose of DNA transferred was 1.6 µg. The transfection activities of CS-DNA-PEG were preserved and a higher dose (2.4 µg) of pDNA was transferred. This indicated that the transfection efficiency of the PEGylated complexes had been improved. In vivo experiments also showed that CS-DNA-PEG complexes mediated higher gene expression in tissues than did CS-DNA complexes, and that gene expression in tumours induced by CS-DNA-PEG complexes was the highest of all. These results suggested that PEGylation of CS-DNA complexes improves non-viral gene delivery in vitro or in vivo and has the potential to deliver therapeutic genes directly into hepatoma tissues.
High Affinity Pegylated Polyacridine Peptide Polyplexes Mediate Potent in Vivo Gene Expression
NIH Public Access, 2013
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-PEG 5kDa stabilizes a 1 μg pGL3 dose for up to 1 hr in the circulation, resulting in HDstimulated (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.
Journal of controlled release : official journal of the Controlled Release Society, 2014
Non-viral vector formulations comprise typically complexes of nucleic acids with cationic polymers or lipids. However, for in vivo applications cationic formulations suffer from problems of poor tissue penetration, non-specific binding to cells, interaction with serum proteins and cell adhesion molecules and can lead to inflammatory responses. Anionic formulations may provide a solution to these problems but they have not been developed to the same extent as cationic formulations due to difficulties of nucleic acid packaging and poor transfection efficiency. We have developed novel PEGylated, anionic nanocomplexes containing cationic targeting peptides that act as a bridge between PEGylated anionic liposomes and plasmid DNA. At optimized ratios, the components self-assemble into anionic nanocomplexes with a high packaging efficiency of plasmid DNA. Anionic PEGylated nanocomplexes were resistant to aggregation in serum and transfected cells with a far higher degree of receptor-target...
Journal of Biotechnology, 2007
The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.