Mitochondria are central to cellular energy production, apoptosis, and metabolism. Their dysfunction is a hallmark of diseases such as cancer, where they support tumor growth, survival, and resistance to therapy. Consequently, targeting mitochondria represents a promising approach for improving cancer treatment specificity and efficacy. However, achieving efficient and selective mitochondrial delivery remains a key challenge. This Ph.D. thesis, presented as a compendium of scientific articles, explores the rational design of polypeptide-based nanocarriers for mitochondrial targeting in cancer therapy. Polypeptides offer tunability and biodegradability, making them ideal candidates for creating delivery platforms that enhance uptake and specificity. The work particularly focuses on applications in triple-negative breast cancer (TNBC), an aggressive and hard-to-treat cancer type. A comprehensive review of mitochondrial targeting strategies using nanomedicine is included in the introductory annex of the thesis. The first article introduces poly-L-ornithine–poly-L-proline (PLOn-PLPm) diblock copolymers designed to bypass traditional, energy-dependent uptake pathways. At an optimized ratio (1:3), these nanocarriers—referred to as mitochondrial targeting cell-penetrating oligomers (mt-CPOs)—accumulate efficiently in mitochondria, even under depolarized conditions. In MDA-MB-231 TNBC cells, mt-CPOs induced mitochondrial dysfunction and antitumor activity at low concentrations (>IC15). Biophysical studies on model membranes revealed cardiolipin-specific interactions and membrane remodeling essential for mitochondrial targeting. Functionalization with polyglutamic acid (PGA) demonstrated their adaptability for drug delivery systems. Conjugation with Lonidamine and α-tocopherol succinate preserved mitochondrial targeting and improved anti-tumor synergy. In the second article, mt-CPOs were further developed by conjugating light-activated molecular motors (MMs) to the PGA-functionalized PLO6-PLP22 carrier. These P-MM nanostructures retained light-driven motion and transitioned from micelles to worm-like assemblies, enhancing cellular uptake while maintaining mitochondrial localization. The PGA layer improved biocompatibility, reducing cytotoxicity while preserving biological function, highlighting the potential of combining nanomechanical motion with targeted delivery. The third article presents C-TRV3-A, a polypeptide-based nanoconjugate combining mitochondrial targeting and non-viral gene delivery. Built from a poly-L-ornithine backbone linked to TRV3 via a redox-sensitive disulfide bond, this system includes triphenylphosphonium for mitochondrial localization, fluorescent dyes for imaging, and enables triggered intracellular release. When formulated with plasmid DNA and a polyanionic polypeptide (VLC-3), C-TRV3-A polyplexes achieved high transfection efficiency in both 2D and 3D TNBC cell models with minimal toxicity. The formulation also demonstrated stability in human plasma, supporting its potential for gene therapy applications. Together, these studies offer innovative mitochondrial-targeted strategies using polypeptide-based platforms with intrinsic mitochondrial affinity, adaptable functionalization, light-responsiveness, and capacity for gene delivery. These systems lay the foundation for advancing precision nanomedicine and hold promise for future clinical translation in multimodal cancer therapies.
