Carrese, Barbara (2023) Chemo-photothermal and photodynamic therapies for cancer treatment. [Tesi di dottorato]

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Abstract

The integration of early diagnosis and targeted therapy is one of the most promising strategies to fight cancer. The word “theranostics” refers to an approach that combines diagnosis and therapy and exploits, as carrier for contrast agents and drugs, nanoparticles. Recent studies combine photoacoustic imaging with chemotherapeutic drugs (e.g., doxorubicin or metallodrugs), and/or phototherapy. The first aim of this study was to test whether nanoparticles with photoacoustic properties, loaded with doxorubicin, have a higher cytotoxic effect than the free drug, thanks to the escape from multi-drug resistance and to their photothermal proprieties. The second aim of this study was to characterize the properties of two ruthenium (II) complexes (DHQ and -CARB) to be used in photodynamic therapy. The interaction between doxorubicin to HSA-NPs and the drug pH-dependent release were evaluated by fluorometric analysis. The uptake of HSA-NPs and doxorubicin delivered by NPs inside HS578T cells, and inside HS578T spheroids was assessed by using confocal microscopy. To evaluate the morphology changes of spheroids treated with DOX@NPs, images were collected by using light microscopy. The chemo- and photo-thermal efficiency of DOX@NPs, as well as the cytotoxicity of both Ru(II) complexes were analyzed by measuring ATP levels and esterases activity. pDNA cleavage, due to DHQ and -CARB, was analyzed by agarose gel electrophoresis. To assess the death mechanism induced both by DHQ and -CARB complexes, caspases 3/7 and 9 activation, and H₂O₂ cellular increase were analyzed. Results showed that maximum bond capability of doxorubicin to HSA-NPs is in a ratio of 40:1 and that doxorubicin is more released at acid pH than at physiological pH. Moreover, a high level of doxorubicin delivered by NPs inside cell nuclei and a higher toxicity of doxorubicin loaded NPs compared to free doxorubicin were observed. Owing to a synergistic effect, this toxicity increases when doxorubicin delivered by NPs and photothermal laser irradiation at 808 nm wavelength are simultaneously used. In 3D cultures, albumin allows a higher penetration of NPs in HS578T spheroids compared to bare NPs. Furthermore, drug carried thanks to NPs internalization is time-dependent, and doxorubicin delivered by NPs causes a good cytotoxicity. This occurrence is probably due to a SPARC mediated internalization, which allows the multi-drug resistance overcoming, and to a drug release in spheroid acidic microenvironment. Regarding DHQ and -CARB ruthenium (II) complexes, they show a general red shift of the absorption and emission maxima and induce DNA dagame by cutting. These complexes have a higher dose-dependent toxicity and caspases activity after laser irradiation compared to dark conditions, and an increased ROS production compared to control in both HS578T and A375 cell lines. In conclusion, all data obtained from this work highlight that the combined effect of chemo- and photodynamic or photothermal- therapy, thanks to their better efficacy, represents an interesting approach to reduce exposure time of treatment and to decrease the effective dose of the chemotherapeutic agents.

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