Construction of mitochondrial-targeted ferritin carrier: subunit-terminal fused mitochondrial presequences facilitate efficient delivery

Introduction: Protein sorting within mitochondria is intricately linked to the amino acid sequence facilitating the transmembrane transport of proteins into this organelle. Leveraging the Mitochondrial Targeting Signal (MTS)-mediated protein sorting mechanism presents a promising strategy for directing therapeutic agents into the mitochondria.

Objectives: By fusing MTS to the subunit terminus of recombinant heavy chain ferritin (HFn), we aim to establish a highly effective mitochondrial targeting vector. This fusion is designed to enhance the ability to specifically direct and accumulate within mitochondria, and to precisely deliver Lonidamine (LND), a selective metabolic inhibitor, into these organelles, ultimately realizing potent anti-tumor activity.

Methods: Utilizing gene engineering strategies, a plasmid encoding MTS-modified HFn (MTS-HFn) was transferred into E.coli to induce protein expression. At the cellular level, the mitochondrial targeting capacity of MTS-HFn was investigated. Subsequently, LND was encapsulated within MTS-HFn, and its tumoral accumulation and anti-tumor efficacy were studied in tumor-bearing models.

Results: MTS-HFn demonstrated exceptional mitochondrial targeting, achieving a 2.7-fold higher accumulation in mitochondria compared to wild-type HFn. The targeting mechanism exploration unveiled that the positive charge of MTS drives aggregation of HFn around mitochondria, and mediates its entry into the mitochondrial matrix via the TOM/TIM complex. In vivo antitumor activity studies revealed that MTS-HFn preserved its inherent tumor targeting ability and significantly enhanced the tumor suppressive effect of LND, yielding an inhibition rate of 51.06%.

Conclusion: This vector inspired by natural mitochondrial protein sorting represents an optimal hierarchical delivery system for targeting both tumor and mitochondrial, offering a dependable alternative for precise treatment strategies in mitochondrial diseases.

Hierarchical delivery; Human ferritin nanocage; Mitochondria targeting; Mitochondrial targeting signal.