Mitochondria targeted polypeptide-drug conjugate delivery platforms
- Autores: Camilla Pegoraro Pegoraro
- Directores de la Tesis: María J. Vicent Docon (dir. tes.), Inmaculada Conejos Sánchez (codir. tes.), Fernando González Candelas (tut. tes.)
- Lectura: En la Universitat de València ( España ) en 2025
- Idioma: español
- Número de páginas: 371
- Tribunal Calificador de la Tesis: ANA Jesús GARCIA SAEZ (presid.), María de la Fuente Freire (secret.), Arwyn Jones (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Mitochondria are essential organelles central to cellular energy metabolism, apoptosis, and homeostasis. Their unique double-membrane structure and circular DNA enable critical processes such as oxidative phosphorylation (OXPHOS) and the generation of the mitochondrial membrane potential (MMP). This electrochemical gradient drives ATP production, regulates ion balance, facilitates metabolite transport, and orchestrates cellular signaling, establishing mitochondria as indispensable hubs of cellular function.
Beyond energy production, mitochondria play pivotal roles in intracellular signaling and metabolic adaptation, which are critical for both normal physiology and pathological conditions. Their endosymbiotic origin has endowed them with unique features, such as the production of reactive oxygen species (ROS). At physiological levels, ROS act as signaling molecules, regulating cellular processes. However, dysregulated ROS levels can induce oxidative stress and damage, contributing to various diseases, including cancer. In this context, mitochondria become both a driver of disease progression and a therapeutic vulnerability.
In triple-negative breast cancer (TNBC), an aggressive and heterogeneous subtype, mitochondrial adaptations are particularly pronounced. Tumor cells rely on mitochondrial dynamics, including fission and fusion, and a functional balance between glycolysis and OXPHOS to meet their high energy and biosynthetic demands. Elevated ROS levels further highlight mitochondria's dual role in TNBC, facilitating tumor survival while presenting exploitable therapeutic targets. However, the selective delivery of therapeutic agents to mitochondria remains a challenge due to biological barriers such as cellular uptake, endosomal escape, and subcellular localization.
Nanomedicine has emerged as a promising strategy to address these barriers. Polymer-based systems, particularly those using polypeptides, offer distinct advantages such as biocompatibility, biodegradability, and modular design. These platforms can be engineered to exploit mitochondrial features, including their high MMP and cardiolipin-rich membranes, enabling precise targeting with minimal off-target effects. Furthermore, polypeptide-based systems can integrate advanced functionalities, such as controlled drug release triggered by tumor-specific conditions like elevated ROS or acidic pH. This versatility allows the combination of multiple therapeutic approaches, including chemotherapy and gene therapy, within a single delivery platform, advancing precision medicine in oncology.
This thesis, structured as a compendium of publications, aims to advance subcellular therapy through the development of polypeptide-based polymer therapeutics targeting mitochondria, with a particular focus on TNBC. It addresses critical challenges such as overcoming biological barriers, achieving precise subcellular localization, and validating therapeutic efficacy in physiologically relevant models.
The first study focused on designing diblock copolymers composed of poly-L-ornithine (PLO) and poly-L-proline (PLP), termed PLOn-PLPm, to achieve selective mitochondrial targeting. PLO polycationic nature facilitated cellular uptake, while PLP was functionalized to promote mitochondrial localization through potential- and concentration-dependent mechanisms. A library of PLOn-PLPm copolymers was synthesized using N-carboxyanhydride ring-opening polymerization (NCA-ROP), with variations in block ratios, chain lengths, and polycationic block types (e.g., polylysine and polyarginine). Among these, copolymers with a 1:3 block ratio consistently demonstrated energy-independent cellular uptake, selective mitochondrial accumulation, and potent activity in TNBC cells. Mechanistic studies revealed specific interactions with cardiolipin (CL)-rich mitochondrial membranes, leading to membrane remodeling and functional disruptions, including ROS production, mitochondrial depolarization, and OXPHOS inhibition at sub-IC50 concentrations. Functionalization with a polyglutamic acid (PGA) backbone further enhanced biocompatibility, reducing toxicity and enabling scalable synthesis. Additionally, conjugating PLO6-PLP22 with therapeutic agents like lonidamine (Loni) and alpha-tocopherol succinate (alpha-TOS) significantly enhanced mitochondrial localization and cytotoxicity by disrupting mitochondrial metabolism, achieving substantial reductions in IC50 values.
The second study expanded the application of polypeptide platforms by integrating light-activated molecular motors (MMs). A propargyl-derivatized MM was conjugated to a PGA-based carrier (PLO6-PLP22 sidechains) via click chemistry, forming the P-MM nanoconjugate. Under 405 nm light irradiation, P-MM underwent a structural transformation from compact micelles to elongated worm-like structures, significantly enhancing cellular uptake in TNBC models without inducing oxidative stress. Mitochondrial targeting remained intact post-irradiation, demonstrating the potential of P-MM for controlled, light-triggered delivery of therapeutic agents. This study highlights the dynamic possibilities of molecular motors in intracellular delivery and establishes a proof-of-concept for their integration into biologically compatible systems.
The third study focused on developing multifunctional nanoconjugates for simultaneous mitochondrial targeting and non-viral gene delivery. The C-TRV3-A nanoconjugate featured a PLO backbone modified with PEG for stability, a triphenylphosphonium (TPP) moiety for mitochondrial targeting, and a redox-sensitive linker for controlled intracellular drug release. After endosomal escape and intracellular reduction, C-TRV3-A localized specifically to mitochondria. For gene delivery, C-TRV3-A was incorporated into polyplexes with plasmid DNA and an anionic shielding polymer, demonstrating high stability, low toxicity, and efficient transfection in both 2D and 3D TNBC models. Notably, in 3D cultures, dose-dependent transfection efficiency reflected the system's ability to overcome structural and penetration barriers, further emphasizing its therapeutic potential in challenging cancer subtypes like TNBC.
In conclusion, this thesis advances subcellular therapy by developing platforms that enhance cytosolic delivery and mitochondrial targeting, providing a strong foundation for preclinical translation. These polypeptide-based systems show significant potential for applications in precision medicine, gene therapy, and targeted drug delivery. Future directions include optimizing pharmacokinetics, integrating ROS-sensitive linkers for controlled drug release, and expanding in vivo evaluations to further explore their therapeutic impact in TNBC and other disease models.
