Influence of Defined Hydrophilic Blocks within Oligoaminoamide Copolymers: Compaction versus Shielding of pDNA Nanoparticles

Abstract

Cationic polymers are promising components of the versatile platform of non-viral nucleic acid (NA) delivery agents. For a successful gene delivery system, these NA vehicles need to comprise several functionalities. This work focuses on the modification of oligoaminoamide carriers with hydrophilic oligomer blocks mediating nanoparticle shielding potential, which is necessary to prevent aggregation or dissociation of NA polyplexes in vitro, and hinder opsonization with blood components in vivo. Herein, the shielding agent polyethylene glycol (PEG) in three defined lengths (12, 24, or 48 oxyethylene repeats) is compared with two peptidic shielding blocks composed of four or eight repeats of sequential proline-alanine-serine (PAS). With both types of shielding agents, we found opposing effects of the length of hydrophilic segments on shielding and compaction of formed plasmid DNA (pDNA) nanoparticles. Two-arm oligoaminoamides with 37 cationizable nitrogens linked to 12 oxyethylene units or four PAS repeats resulted in very compact 40-50 nm pDNA nanoparticles, whereas longer shielding molecules destabilize the investigated polyplexes. Thus, the balance between sufficiently shielded but still compact and stable particles can be considered a critical optimization parameter for non-viral nucleic acid vehicles based on hydrophilic-cationic block oligomers.

EGFR Targeting and Shielding of pDNA Lipopolyplexes via Bivalent Attachment of a Sequence‐Defined PEG Agent

“…To the same samples 250 IU of heparine sulfate was added to determine anionic stress tolerance (Figure S6C, Supporting Information). Here we found, as reported previously, that LPEI polyplexes are very sensitive, leading to 100% EtBr fluorescence corresponding to full pDNA release. These effects were minor pronounced for LPEI–PEG 2k –GE11, retaining ≈13% pDNA, nevertheless they were notably increased in comparison to 454‐ based polyplexes, where only 70% could be released.…”

“…Also pre‐PEGylated polyplexes, consisting of targeted or untargeted two‐arm‐His‐PEG 24 and 200 ng of pDNA, showed complete pDNA retention (Figure S3A, Supporting Information) at N/P 12. It was reported that lower N/P ratios for these kinds of carriers were sufficient to completely bind pDNA . For reasons of comparability between post‐PEGylated lipopolyplexes and pre‐PEGylated two‐arm polyplexes, all further experiments were conducted with N/P 12.…”

A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

“…There is evidence that integration of PEG chains into electrostatically formed nanoparticles decreases its stability (Morys et al, 2017). On the one hand, this could be a critical problem if the polyplex disintegrates before it delivers its payload.…”

“…Generally, azide-bearing core oligomers were modified with PEG-ligands 45 min after polyplex formation (Fig. 6B) because coupling PEG-ligands to core oligomers before polyplex formation hampers siRNA compaction (Morys et al, 2017). This method has already been established by Klein et al (2018) and was used here to validate results generated with core polyplexes that had PEG-ligands attached by lipid anchors.…”

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“…Generally, azide-bearing core oligomers were modified with PEG-ligands 45 min after polyplex formation (Fig. 1D) because coupling PEG-ligands to core oligomers before polyplex formation hampers siRNA compaction (Morys et al, 2017). This method has already been established by Klein et al (Klein et al, 2018) and was used here to validate results generated with core polyplexes that had PEG-ligands attached by lipid anchors.…”