Functional block copolymers have led to development of electrostatically self-assembleed polyplex micelles (PMs) as gene delivery carriers for efficient gene therapy. Major demands for achieving efficient gene transfection using PMs are (i) maintenance of loaded pDNA strongly during delivery process, (ii) endosomal escapability, and (iii) release of loaded pDNA in cytoplasm for efficient transcription. To overcome these three barriers, we focused on phenylboronate ester between phenylboronic acid (PBA) and diol compounds, which can be cleaved in response to endosomal pH and competitive diol compounds, including adenosine triphosphate (ATP) presenting at high concentration in cytoplasm. As block copolymers, poly(ethylene glycol)-poly{N’-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-PAsp(DET)) was used because PEG-PAsp(DET) is one of the appealing block copolymers to elicit the endosomal escapability. In this design, we expected the enhancement of PMs stability in the extracellular condition by the phenylboronate ester cross-linking, the efficient endosomal escape by PEG-PAsp(DET) and the release of loaded pDNA by the cleavage of the cross-linking in response to cytosolic ATP concentration (Fig. 1). First, PBA and N-substituted gluconamide (GlcAm) as a polyol compound were respectively introduced to PAsp(DET) side chain. The introduction ratios of PBA and the GlcAm were varied to find the optimized combination in terms of stability and responsivity to ATP. The PMs prepared from pDNA and the combination of optimized two block copolymers elicited significant tolerability against polyanion attack. Moreover, the cross-linked PMs exhibited the release of loaded pDNA under intracellular ATP concentration. In the cell-based experiments, the cross-linked PMs showed improved cellular uptake and also transfection efficiency compared to that of non-cross-linked PMs. In addition, the cross-linked PMs showed efficient endosomal escapability and ATP-responsive loaded pDNA release in cells. These results indicated that the introduction of the pH and ATP dual responsive PBA/diol cross-linking in the PM core was a promising approach for improving efficiency of gene delivery system.

Design of polyplex micelles with phenylboronate ester cross-linking in the core exerting promoted gene transfection through responsivity to intracellular pH and ATP concentration

256th National Meeting (2018)

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