UNRAVELING NANOBIOINTERACTION MECHANISMS FOR NANOMEDICINE APPLICATIONS: HOW INTRA- AND EXTRA-CELLULAR MICROENVIRONMENTS SHAPE CELL RESPONSE TO NANOMATERIALS

NANOMEDICINE AIMS TO REPLACE TRADITIONAL DRUGS WITH ACTIVE NANOSCALE TOOLS THAT INTERACT WITH BIOLOGICAL SYSTEMS IN A TARGETED MANNER. ALTHOUGH MANY NANOMATERIALS ARE BEING EXPLORED, MOST REMAIN AT EARLY STAGE AND THEIR SUCCESS DEPENDS ON HOW CELLS “SENSE” AND PROCESS THESE MATERIALS. UNDERSTANDING NANOBIOINTERACTION MECHANISMS IS ESSENTIAL TO DESIGN SAFER, MORE EFFECTIVE NANOMEDICINES AND IS THE FOCUS OF THIS THESIS, WHICH ADDRESSES THEM AT THREE LEVELS: INTRACELLULAR ANTIOXIDANT MECHANISMS, ORGANELLE CROSSTALK AND MICROENVIRONMENT-DRIVEN IMMUNE MODULATION. THE FIRST PART INVESTIGATES HOW PLATINUM NANOPARTICLES (PTNPS), ACTING AS ANTIOXIDANT NANOZYMES BUT CONFINED INTO ENDO-LYSOSOMAL COMPARTMENTS, PROTECT HEPG2 CELLS FROM OXIDATIVE STRESS INDUCED BY DICHLORODIPHENYLETHYLENE (DDE). PTNPS PRESERVE CELL VIABILITY AND MORPHOLOGY, SUPPRESS DDE-DRIVEN ROS ACCUMULATION AND RESTORE MITOCHONDRIAL HOMEOSTASIS BY ENHANCING ANTIOXIDANT DEFENCES, PROMOTING FUSION-BIASED MITOCHONDRIAL DYNAMICS AND LIMITING APOPTOSIS. ULTRASTRUCTURAL ANALYSIS REVEALS APPOSITION BETWEEN PTNP-CONTAINING ENDO-LYSOSOMES AND MITOCHONDRIA, SUGGESTING THAT INTER-ORGANELLE CONTACT SITES CONTRIBUTE TO THIS INDIRECT MITOCHONDRIAL RESCUE. THE SECOND PART EXPLORES THE MECHANISM OF ACTION OF PTNPS BY COMPARING THEM WITH SIZE-MATCHED GOLD NANOPARTICLES (AUNPS) LACKING INTRINSIC ANTIOXIDANT ACTIVITY. IN HEPG2 CELLS, BOTH NANOMATERIALS LOCALIZE INTO ENDO-LYSOSOMES, REMODEL THE ENDOCYTIC PATHWAY AND UPREGULATE MITOCHONDRIAL PROTEINS SOD2 AND MFN2 WITHOUT CHANGES IN MITOCHONDRIAL MORPHOLOGY, INDICATING COMPARABLE CELL UPTAKE AND ADAPTATION. HOWEVER, ONLY PTNPS REDUCE ROS IN H₂O₂- AND LPS-INDUCED STRESS MODELS. PROXIMITY LIGATION ASSAY AND ULTRASTRUCTURAL ANALYSES SHOW THAT PTNPS, BUT NOT AUNPS, INCREASE MITOCHONDRIA–LYSOSOME AND ER–LYSOSOME CONTACTS WHILE REDUCING ER–MITOCHONDRIA CONTACTS, WITHOUT AFFECTING LYSOSOMAL ACIDITY. FURTHERMORE, INTEGRATED LIPIDOMIC AND PROTEOMIC ANALYSES DEMONSTRATE THAT BOTH PT AND AU NP-INDUCED TRIACYLGLYCEROL (TAG) DEPLETION AND A REORGANIZATION OF MITOCHONDRIA–LYSOSOME PROTEOMES IN A NANOPARTICLE-DEPENDENT MANNER. TAKEN TOGETHER, THESE DATA INDICATE THAT LYSOSOMAL PTNPS MAY ACT AS CATALYTIC HUBS THAT REWIRE ORGANELLE COMMUNICATION. THE FINAL PART EXAMINES HOW ECM-LIKE MICROENVIRONMENTS INFLUENCE CELLULAR RESPONSES TO NANOMATERIALS THAT ARE NOT SIMPLY DISPERSED IN BIOLOGICAL FLUIDS BUT IMMOBILIZED WITHIN A MATRIX. WE FOCUSED ON MACROPHAGES BECAUSE THEY ARE AMONG THE FIRST INNATE IMMUNE CELLS TO RESPOND TO FOREIGN AGENTS. TO THIS END, THP-1–DERIVED HUMAN MACROPHAGES ARE CULTURED ON GELATIN SCAFFOLDS FUNCTIONALIZED WITH MULTIWALLED CARBON NANOTUBES (MWCNTS). IN THIS ECM-MIMETIC 3D SETTING, MATRIX-EMBEDDED MWCNTS MAINTAIN CELL VIABILITY BUT INDUCE MORPHOLOGICAL CHANGES AFTER 48 HOURS. MACROPHAGES REMODEL THE MATRIX, INTERNALIZE EXPOSED FIBRES, INCREASE ROS AT 24 H AND DISPLAY A SELECTIVE RISE IN CD80⁺ CELLS WITHOUT CD206 INDUCTION AT 48 H, CONSISTENT WITH CONTROLLED M1-BIASED POLARIZATION. THEY ALSO PRODUCE A SECRETOME THAT MARKEDLY RESTRAINS GLIOBLASTOMA SPHEROID GROWTH, SHOWING THAT ECM-EMBEDDED CNTS CAN DRIVE A PARACRINE ANTI-TUMOUR RESPONSE. TOGETHER, THESE FINDINGS SHOW PHYSICOCHEMICAL AND CATALYTIC PROPERTIES OF NANOMATERIALS, AS WELL AS THE EXTRACELLULAR MICROENVIRONMENT, MODULATE ORGANELLE FUNCTION, MEMBRANE CONTACT SITE DYNAMICS AND IMMUNE RESPONSES. A MAJOR CHALLENGE IS TO HARNESS THESE PROPERTIES TO CONTROL MEMBRANE CONTACT SITE (MCS) SIGNALLING AND IDENTIFY MOLECULAR TARGETS FOR NANOMATERIALS THAT TUNE THESE PROCESSES AND ENABLE THERAPIES FOR DISEASES LINKED TO ABERRANT MCS, AN AREA THAT REMAINS UNDEREXPLORED. FINALLY, THE 3D CNT–GELATIN PLATFORM OFFERS A TOOL TO STUDY HOW MATRIX ARCHITECTURE SHAPES IMMUNE RESPONSES AND A BASIS FOR DESIGNING BIOMIMETIC SCAFFOLDS THAT STEER CELLULAR AND IMMUNE BEHAVIOUR IN CANCER.