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Biofunctional upconversion nanoparticles for cancer theranostics
thesisposted on 2022-03-28, 17:00 authored by Liuen Liang
Development of new approaches for diagnosis and therapy of tumours, termed theranostics, is one of the most dynamic areas of the life sciences, where new nanomaterials afford new opportunities. The nanomaterial merits include programmability of their physical and chemical properties; abundance of reactive functional groups on their surface; large effective surface area and optimal size, which determines preferential accumulation of nanoparticles (NPs) in tumour tissue.Photoluminescent nanomaterials add to the list of merits their high optical contrast achievable on the crowded background of biological cells, tissues and whole organisms.In order to fully harness the potential of photoluminescent NPs, they need to be coupled with functional biological molecules, accomplished by procedures of NP surface redressing and bioconjugation, to enable controlled targeting and therapeutic action in the delicate environment of a biological system. Interactions of these biofunctional hybrid assemblies with live cells and organisms, however, are overly complex, poorly understood and controlled. It is increasingly being accepted that NPs encountering biological medium are swiftly coated by a biomolecular adsorption layer called “protein corona”. Consequently, the molecular machinery of a living cell or organism will interact with the corona rather than biofunctional hybrid assemblies, making it a key determinant of the biological response of NP exposure. In this PhD thesis, the design and characterisation of photoluminescent biofunctional nanoparticles and their applications for targeted delivery and photodynamic therapy of cancer cells are addressed, in addition to the systematic investigation of protein corona formation on polymer-coated nanoparticles. My thesis research was centred at a new-generation of biofunctional photoluminescent nanoparticles with unique optical properties termed upconversion nanoparticles (UCNPs). UCNPs are photoexcited by near-infrared light (NIR) at 980 nm capable for deep penetration (up to 1 cm) in biological tissue. This excitation confers another key advantage of background-free imaging in cells and tissues due to very little excitation of autofluorescence. UCNP emission is tuneable by design from ultraviolet to visible or even NIR spectral bands, which enabled my colleagues and me to address the main limitation of photodynamic therapy (PDT), shallow treatment depths. Indeed, the conversion of deeply-penetrating near-infrared to visible light allows photosensitisation of such potent PDT agents, as fluorescent protein KillerRed and Rose Bengal. In the first paper, we have demonstrated the PDT therapeutic efficacy of a water-soluble photosensitiser protein Killer Red coupled to UCNP covalently. The spectral overlap between the UCNP emission and KillerRed absorption ensured the efficient energy transfer, leading to reactive oxygen species (ROS) generation upon the excitation at 980nm, which exerted toxicity to MDA-MB-231 breast cancer cells. Cross-comparison between the conventional KillerRed and UCNP-mediated KillerRed PDT treatment of cancer cells buried under 1-cm muscle tissue clearly demonstrated superiority of KillerRed-UCNP photosensitisation by the NIR light, with no detectable PDT effect in the case of KillerRed photosensitised by a yellow laser. In the second paper, we have reported a facile strategy to assemble PDT nanocomposites functionalised for cancer targeting, based on the coating of UCNP with a silica layer encapsulating the Rose Bengal photosensitiser and bioconjugation to antibodies through a bifunctional fusion protein consisting of a solid-binding peptide linker (L) genetically fused to Streptococcus Protein G’ (PG). The fusion protein (Linker-Protein G, LPG) mediates the functionalisation of silica-coated UCNPs with cancer cell antibodies allowing for specific target recognition and delivery. The resulting nanocomposites were shown to target cancer cells specifically, generate intracellular reactive oxygen species under 980-nm excitation, and induce NIR-triggered phototoxicity to suppress cancer cell growth in vitro. In the third paper, the effect of protein corona formation on UCNPs coated with positively- (polyethylenimine), negatively- (polyacrylic acid) charged and nearly neutral (polyethylene glycol) polymers was investigated. Our protein assaying study corroborated the TEM observation, showing profound effect of the UCNP-polymer surface charge: UCNP-polyethylenimine acquired four-fold of protein content as compared to the other coatings. The composition of protein binding to UCNP is notably influenced by the surface charges of the UCNP-polymer. We also found that the protein corona inhibited the cell binding and cellular uptake of UCNPs coated with positive and neutral polymers, whereas enhanced that of UCNP-polyethylenimine. Moreover, the presence of protein corona slightly mitigated the mild cytotoxic effects of nanoparticles. These results provide valuable guidance into rational design of UCNP-based biofunctional agents. In a separated published work carried out with my colleagues, we investigated effect of the surface functionalisation of UCNPs on the blood circulation lifetime using in vivo early-stage developed animal model (chicken embryo). The preference of NP coating with polyethylene glycol was expected and confirmed. This study was critical for purpose-design of NPs evading rapid filtration from the blood circulation, therefore maximising nanoparticle accumulation in the tumour in vivo.