Monomolecular Assembly of siRNA and Poly(ethylene glycol)−Peptide Copolymers

Abstract

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.

Nanosized Multifunctional Polyplexes for Receptor-Mediated SiRNA Delivery

“…The Dde protecting group was removed by washing the resin with 2% hydrazine in DMF until completion of cleavage was confirmed by lack of absorption at 290 nm. At the free amine the synthesis was continued, adding first a defined Fmoc-PEG 24 -OH (QuantaBiodesign, Powell, OH, USA) building block followed by Fmoc-Glu-OtBu and finally N 10 -(trifluoroacetyl)pteroic acid. The tfa-protecting group was removed using 1 M ammonium hydroxide/DMF, 1:1 (v/v).…”

“…For the distribution analysis of the polyplexes FolA-PEG 24 -K(Stp 4 -C) 2 , FolA-PEG 48 -K(Stp 4 -C) 2 , or FolA-PEG 72 -K(Stp 4 -C) 2 each containing Cy7-labeled siAHA1 (sense: 5 0 -(Cy7)(NHC 6 )-GGAuGAAGuGGAGAuuAGudTsdT-3 0 ; antisense: 5 0 -ACuAAUCUCcACUUcAUCCdTsdT-3 0 ) were injected intravenously. Fluorescence measurement by a CCD camera was started immediately after injection and repeated after 0.25, 1, 4, and 24 h. To demonstrate the functionality of receptor targeting in vivo FolA-PEG 24 -K(Stp 4 -C) 2 containing siAHA1-Cy7, A-PEG 24 -K(Stp 4 -C) 2 containing siAHA1-Cy7, or pure siAHA1-Cy7 was injected intratumorally. Fluorescence signal was measured after 0, 4, 24, 48, 72, 96, and 120 h. Data interpretation was done with equalized color bar scales for each trial.…”

“…As it is known that the coupling of folic acid via its R-carboxy group results in a reduced binding affinity to its receptor, the coupling was performed via its γ-carboxy group. 30 Purity and identity of the synthesized structure FolA-PEG 24 Design and Synthesis of the Endosomolytic Active siRNAÀ Influenza Peptide Conjugate. As the targeted polycationic peptide includes functional substructures for siRNA binding, polyplex stability, and folate receptor targeting, endosomal escape is the remaining critical bottleneck.…”

“…Although a clear tumortargeting effect could be shown within this imaging experiment, the intracellular delivery of the siRNA and thus the functionality of the carrier had to be evaluated in a target gene silencing experiment. For this purpose EG5-Inf7 siRNA was injected intratumoraly, using FolA-PEG 24 -K(Stp 4 -C) 2 (356) as carrier. In stained tumor slices of tumors harvested 24 h after treatment the formation of asters could be detected (Figure 5b).…”

Gene therapy progress and prospects: synthetic polymer-based systems

“…This modulated the structure of the resulting polyplexes, ranging from extended ring-like structures to highly compact toroidal structures. Importantly, stable single-polynucleotide complexes could be generated, as described similarly for sequence-defined synthetic peptidebased block copolymers by DeRouchey et al 31 or previous published work for synthetic polylysine-PEG conjugates.…”