Utilization of retro-inverso peptide ligand of Transferrin receptor for enhanced polymer-based liver-targeted gene delivery

다국어 초록 (Multilingual Abstract)

Liver-targeted gene therapy offers great promise for the treatment of hepatic disorders; however, its clinical progress has been hindered by the lack of vectors that can ensure both safety and high delivery efficiency. The 25 kDa branched polyethylenimine (BPEI) polymer has long been recognized as an effective non-viral vehicle because of its remarkable buffering capacity and ability to facilitate endosomal escape. Nevertheless, its non-selective interactions with biological components often result in systemic toxicity, restricting its use in therapeutic applications. To overcome these drawbacks, a hepatocyte-specific carrier system was designed by covalently linking BPEI25K to peptides that mimic the transferrin receptor (TfR) ligand, taking advantage of the receptor’s elevated expression on liver cells. Two types of peptides were employed for this purpose: the native L-form peptide (HAIYPRH) and a retro-inverso D-form variant (HRPYIAH) that exhibits improved resistance to enzymatic degradation. These conjugates, referred to as BPEI–TfRL and BPEI–TfRD, were synthesized and complexed with plasmid DNA (pDNA) to form nanoscale polyplexes. Both polymer conjugates efficiently condensed pDNA into stable nanoparticles with a positive surface charge at an optimal polymer-to-DNA ratio of 15:1 (w/w). When evaluated in AML-12 murine hepatocytes, the unmodified BPEI showed minimal gene transfer capability. In contrast, both TfR-modified carriers enhanced transfection outcomes, with BPEI–TfRD producing the strongest gene expression. This was evident from pronounced GFP fluorescence observed at 24 h and 48 h post-transfection and from a significant elevation in S1P1R mRNA levels. Overall, these findings suggest that the D-form peptide-conjugated BPEI platform holds substantial potential as a selective and efficient vector for liver-specific gene therapy.

목차 (Table of Contents)

• Abstract 1
• Introduction 4
• Material and Methods 15
• Results 22
• Discussion 57
• Conclusion 61
• References 63