Reversible Stabilization of Nanofiber-Polyplexes through Introducing Cross-Linkages.

Non-viral gene delivery systems are typically designed vector systems with contradictory properties, namely sufficient stability before cellular uptake and instability to ensure the release of nucleic acid cargoes in the transcription process after being taken up into cells. We reported previously that poly-(L-lysine) terminally bearing a multi-arm PEG (maPEG-PLL) formed nanofiber-polyplexes that suppressed excessive DNA condensation via steric repulsion among maPEGs and exhibited effective transcriptional capability in PCR amplification experiments and a cell-free gene expression system. In this study, the reversible stabilization of a nanofiber-polyplex without impairing the effective transcriptional capability was investigated by introducing cross-links between the PLL side chains within the polyplex using a cross-linking reagent with disulfide (SS) bonds that can be disrupted under reducing conditions. In the presence of dextran sulfate and/or dithiothreitol, the stability of the polyplex and the reactivity of the pDNA were evaluated using agarose gel electrophoresis and real-time PCR. We succeeded in reversibly stabilizing nanofiber-polyplexes using dithiobis (succinimidyl propionate) (DSP) as the cross-linking reagent. The effect of the reversible stabilization was confirmed in experiments using cultured cells, and the DSP-crosslinked polyplexes exhibited gene expression superior to that of polyethyleneimine polyplexes, which are typical polyplexes.

Gene expression of ternary complexes through the compaction of nanofiber-polyplexes by mixing with lipofectamine.

For the development of an effective nonviral gene vector, ternary complexes were prepared through the compaction of nanofiber-polyplexes. These were formed using pDNA and a head-tail type polycation bearing a multi-arm poly(ethylene glycol) head and a poly(l-lysine) tail, and this strategy was based on the crowding effect of poly(ethylene glycol) in the polyplex. Mixing was carried out using a cationic lipid (lipofectamine), which is a commercially available transfection reagent. Through ternary complex formation, the elongated morphology of nanofiber-polyplexes was found to compact into a spherical shape with an average diameter of ca. 100 nm. Accompanying ternary complex formation, the compaction of the nanofiber-polyplexes can improve cellular uptake and helps the ternary complex to retain its smooth transcription/translation process, which is characteristic of nanofiber-polyplexes. As a result, ternary complexes prepared at an optimal mixing ratio exhibit a high transfection efficiency compared with lipofectamine lipoplexes.

Nanofiber Polyplex Formation Based on the Morphology Elongation by the Intrapolyplex PEG Crowding Effect.

We prepared a multiarm poly(ethylene glycol)-poly(l-lysine) block copolymer (maPEG-PLL) with a size-controllable maPEG head and a cationic PLL tail for the evaluation of the effect of maPEG crowding to the polyplex formation with plasmid DNA. maPEG-PLLs of various compositions were synthesized and the formation of a polyplex was confirmed by gel retardation assay. The maPEG-PLL exhibited noncooperative polyplex formation behavior, suggesting the effective hydration of the polyplex. Also, an increase in the size of the maPEG head induces the elongation of polyplex morphology from spherical aggregates to nanorods and nanofibers because of the intrapolyplex PEG crowding effect. Furthermore, an increase in the size of the maPEG head also improves the effective inhibition of the decrease in cell-free gene expression, indicating the importance of the control of pDNA packaging in the polyplex.