Post-PEGylation of siRNA Lipo-oligoamino Amide Polyplexes Using Tetra-glutamylated Folic Acid as Ligand for Receptor-Targeted Delivery

Abstract

For efficient and receptor-specific siRNA delivery, a new post-PEGylation strategy was established to provide siRNA polyplexes with targeting and shielding agents. For this purpose, core nanoparticles were formed by complexing siRNA with sequence-defined cationic lipo-oligomers. The T-shaped bis-oleoyl-oligoethanamino amides 454 and 595, containing stabilizing tyrosine and cysteine residues, were applied. These core nanoparticles were surface-shielded by reaction with maleimido-polyethylene glycol (Mal-PEG) reagents, optionally containing the targeting ligand folic acid (FolA). The PEGylation had two unpredicted consequences. First, FolA-PEG surface-modified polyplexes agglomerated due to the hydrophobicity of folic acid, resulting in ligand-independent gene silencing. This problem was solved by the use of tetra-γ-glutamyl folic acid (gE4-FolA) as targeting ligand. Post-PEGylated gE4-FolA siRNA polyplexes displayed sizes of 100-200 nm and mediated receptor specific uptake and effective gene silencing. Second, PEGylation triggered a destabilization of polyplexes, which was uncritical in cell culture but a limiting factor in vivo, as revealed by biodistribution studies in mice. This problem was partially overcome by selecting 595 (containing two CRC stability motifs) for polyplex core formation and an optimized lower degree of gE4-FolA PEGylation reagent. Biodistribution in L1210 tumor bearing mice demonstrated a significantly reduced lung signal and extended persistence of siRNA polyplexes (up to 8 h), with moderate delivery into the tumor. Further polyplex stabilization will be required for effective tumor-targeted delivery.

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

“…For postintegration into core lipopolyplexes, the bivalent structure Cys(Npys) 2 ‐PEG 24 ‐GE11 was found to be the best agent, yielding highly specific, EGFR dependent gene transfer, demonstrated with luciferase marker gene and sodium iodide symporter (NIS) gene expression studies. Previous work already pointed out that postmodification represents a promising tool for shielding and targeting pDNA as well as siRNA polyplexes . The superior effect of the bivalent over monovalent linkage is well consistent with similar findings using multivalent coatings of nucleic acid nanoparticles …”

“…Peripheral tyrosine trimers (Y 3 ) increase polyplex stability via interoligomeral π–π stacking of aromatic rings; C‐terminal as well as N‐terminal cysteine residues provide additional, disulfide triggered polyplex stabilization as well as anchors for the subsequent post‐PEGylation. This oligomer has been extensively investigated as core oligomer for receptor dependent siRNA delivery in vitro as well as in vivo, e.g., via folate receptor, transferrin receptor as well as EGFR, but has been never used as core carrier for receptor targeted pDNA delivery. Pre‐PEGylation of 454 before polyplex formation was unsuccessful, resulting in polyplex aggregation (SM, unpublished results).…”

“…However, covalent modification of cationic carriers with hydrophilic chains may also diminish nanoparticle compaction and other favorable properties . Postintegration of shielding agents into preformed nanoparticles emerged as encouraging alternative to avoid such unfavorable effects . Use of shielding agents with multivalent attachment sites proved to be especially effective, as improved shielding synergizes with lateral stabilization of the nanoparticle by crosslinking the carrier molecules …”

Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes

“…In contrast, in the cells treated with standard siGFP polyplexes, the silencing effect was significantly decreased (45-63% for TCPs and 35% for 356), further highlighting that Inf7 peptide is necessary for sufficient release of siRNA into cytosol, as previously reported earlier [16,35,36]. Besides, recently we verified that the uptake of 100-200 nm FR-targeted polyplexes could be blocked completely by excess free folate, while the agglomerated large particles (>200 nm) might partially undergo receptor-independent uptake and gene silencing [61]. The luciferase activity in KB/eGFPLuc cells treated with controls, including siCtrl polyplexes and siCtrl-Inf7 polyplexes were similar to untreated cells, which suggested that there is no intrinsic cytotoxicity of TCPs.…”

Metabolic stability and persistence of expression of mRNA for nonviral gene delivery

“…The polyplexes formed by crosslinking were hypothesized to be more metabolically stabilized in circulation than the noncrosslinked polyplexes. In addition, post-PEGylation of sulfhydryl crosslinked polyplexes was performed to limit the amount of PEG on the surface of the polyplex because too much PEG could limit transfection efficiency 221 . The C-C(depro) was incubated for 1 hr in a pH neutral buffer with dsmRNA and then PEGylated with 10 eq mPEGmal5kDa.…”