Gene delivery into plant cells provides the basis for many applications in agriculture and plant biotechnology fields. From this point of view, gene carrier systems applicable to plants are gaining interest. Recently, we developed a gene carrier for plants, maleimide-conjugated tetra(ethylene glycol)-block-poly(l-lysine) (MAL-TEG-PLL). MAL-TEG-PLL containing oligo(l-lysine) segments electrostatically interacted with plasmid DNA (pDNA) and formed a micelle that presented maleimide groups on its surface. Through thiol-maleimide coupling reactions, the micelle was successfully postmodified with cationic cell-penetrating peptides (CPP) without attenuating CPP ability, thereby enabling efficient gene delivery into plants. However, current micelle system needs further improvements in terms of gene delivery efficiency. In this study, we aimed to optimize polycation segments for gene carriers and CPPs for micelle postmodification. We designed two gene carriers: MAL-TEG-(KH)14 and MAL-TEG-(RH)14, where (KH)14 and (RH)14 represent fourteen repeats of (l-lysine-l-histidine) and (l-lysine-l-arginine) units, respectively. DLS measurements in buffer solutions (0–20 mM HEPES) revealed that MAL-TEG-(KH)14/pDNA micelle was disrupted at buffer concentration above 10 mM, whereas MAL-TEG-(RH)14/pDNA micelle was stable at the corresponding buffer concentration. Next, by means of the thiol-maleimide coupling, we postmodified both micelles with two CPPs: C-BP100 (CKKLFKKILKYL) and C-KAibA (CKXAKXAKXA, X = Aib). RP-HPLC and DLS analyses indicated the successful preparation of BP100-C-MAL-TEG-(KH)14 (conversion rate of maleimide group, 88 ± 7%; hydrodynamic diameter, 80 ± 1 nm), KAibA-C-MAL-TEG-(KH)14 (89 ± 6%, 94 ± 3 nm), BP100-MAL-TEG-(RH)14 (78 ± 5%, 89 ± 2 nm), and KAibA-C-MAL-TEG-(RH)14 (98 ± 2%, 92 ± 1 nm). We are now evaluating the gene delivery efficiency of these micelles in plants to investigate the effect of the polycation segment and CPP.

Micelles postmodified with cell penetrating peptides for efficient gene delivery in plants

2019 Fall National Meeting

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