Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA vs PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA vs pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems.

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Amphiphilic triblock copolymers represent a versatile delivery platform capable of co-delivery of nucleic acids, drugs, and/or dyes. Multifunctional cationic triblock copolymers based on poly(ethylene glycol), poly-ε-caprolactone, and polyethylene imine, designed for the delivery of siRNA, were evaluated in vitro and in vivo. Moreover, a nucleic acid-unpacking-sensitive imaging technique based on quantum dot-mediated fluorescence resonance energy transfer (QD-FRET) was established. Cell uptake in vitro was measured by flow cytometry, whereas transfection efficiencies of nanocarriers with different hydrophilic block lengths were determined in vitro and in vivo by quantitative real-time PCR. Furthermore, after the proof of concept was demonstrated by fluorescence spectroscopy/microscopy, a prototype FRET pair was established by co-loading QDs and fluorescently labeled siRNA. The hydrophobic copolymer mediated a 5-fold higher cellular uptake and good knockdown efficiency (61 ± 5% in vitro, 55 ± 18% in vivo) compared to its hydrophilic counterpart (13 ± 6% in vitro, 30 ± 17% in vivo), which exhibited poor performance. FRET was demonstrated by UV-induced emission of the acceptor dye. Upon complex dissociation, which was simulated by the addition of heparin, a dose-dependent decrease in FRET efficiency was observed. We believe that in vitro/in vivo correlation of the structure and function of polymeric nanocarriers as well as sensitive imaging functionality for mechanistic investigations are prerequisites for a more rational design of amphiphilic gene carriers.

Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEG-Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation.

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