PhD THESIS DEFENSE: Camilla Pegoraro

PhD Thesis Defense: «Mitochondria targeted polypeptide-drug conjugate delivery platforms».
Camilla Pegoraro.

Abstract

In today’s pharmaceutical era, the success of many emerging therapies depends on their ability to act at the subcellular level. Given the role of mitochondrial dysfunction in diseases such as cancer, there is growing interest in developing systems that can specifically target this organelle. However, delivering therapies to mitochondria remains challenging due to its compartmentalized double-membrane structure and the need to bypass the plasma membrane and ensure cytosolic release. Polypeptide-based systems offer a promising and highly tunable biodegradable platform to overcome these barriers and enable precise mitochondrial delivery. This PhD thesis, developed within the EU Horizon 2020 Innovative Training Network BIOMOLMACS and presented as a compendium of scientific papers, focused on the rational design and evaluation of polypeptide-based nanomedicines for selective mitochondrial targeting in a triple-negative breast cancer (TNBC) model. Two complementary strategies were explored: one based on the development of inherently mitochondria-targeting, cell-penetrating oligomers; the other on hierarchical systems for sequential delivery via endocytosis and mitochondrial localization.

In the first study, we designed poly-L-ornithine (PLO)–poly-L-proline (PLP) diblock copolymers that demonstrated energy-independent uptake and cardiolipin-mediated mitochondrial binding, independent of mitochondrial membrane potential. These systems exhibited potential mitochondria-dependent anti-tumor activity at sub-IC50 levels. Functionalization with polyglutamic acid (PGA) improved biocompatibility while retaining mitochondrial tropism, and conjugation with therapeutic agents such as lonidamine and α-tocopherol succinate further enhanced their mitochondrial accumulation and anticancer potential.

In the second study, we conjugated the PGA-based carrier to a light-driven molecular motor to create a hybrid system capable of enhancing cellular uptake through light-triggered conformational changes, while preserving safety and mitochondrial specificity.

The third study focused on a multifunctional polypeptide-based nanocarrier designed for mitochondrial targeting and non-viral gene delivery. We combined a PLO backbone, polyethylene glycol for stability, a triphenylphosphonium moiety for mitochondrial localization, and a redox-sensitive linker for controlled release. After sequential endosomal escape and intracellular reduction, the system efficiently reached mitochondria. For gene delivery, we formulated polyplexes combining the polycationic platform, plasmid DNA and an anionic shielding polymer, achieving high stability, low toxicity, and efficient transfection in both 2D and 3D TNBC models. In conclusion, this thesis advances subcellular therapeutic strategies by developing polypeptide-based platforms that enhance cytosolic delivery and mitochondrial targeting, providing a solid basis for preclinical translation and future applications in precision medicine.