Compagnin, Chiara (2010) Analisi di effetti biologici indotti dal trattamento in vitro con nanoparticelle per uso terapeutico. [Tesi di dottorato]
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Photodynamic therapy (PDT) is a minimally invasive therapeutic modality mainly used in the oncological field for treating various types of solid tumours. PDT is based on the administration of a photosensitizing agent (PS), that preferentially localizes in neoplastic tissues and generates highly reactive oxygen species (ROS), after activation with appropriate wavelengths of visible light. These species, in particular singlet oxygen (1O2), trigger a cascade of reactions leading to oxidative damage and cell death. One limitation of PDT is represented by the hydrophobic nature of many PS that, favouring aggregation in aqueous medium, strongly depresses the therapeutic efficiency. To overcome this problem and to enhance the efficacy and selectivity of PDT, confining the cytotoxic effects to the surrounding healthy tissues, one can take advantage of the potentialities offered by the emerging nanotechnologies. In oncology, in fact, nanostructures are receiving considerable attention as injectable nanovectors for the selective delivery of drugs to cancer cells and as tumour diagnostic and imaging agents. My PhD project was focused on the use of hydrophilic nanosystems for the delivery of meta-tetra(hydroxyphenyl)chlorin (mTHPC, trade name Foscan®, provided by Biolitec), a hydrophobic second generation photosensitizer characterized by a high antitumoral activity and approved in Europe for the palliative treatment of advanced head and neck cancers. In particular, ORganically Modified SILica nanoparticles (ORMOSIL NP; synthesised in the Department of Chemical Sciences, University of Padova) and liposomes coated with poly(ethylene glycol) (PEG) (Fospeg; provided by Biolitec) were studied as mTHPC delivery systems. The entrapment of mTHPC in NP did not modify its photophysical properties and its efficiency of 1O2 production after irradiation. We evaluated the biological effects of these nanovehicles in human cancer and normal cells comparing dark cytotoxicity, photodynamic efficacy, internalization and subcellular distribution of mTHPC entrapped in NP with respect to mTHPC delivered in standard solvent ethanol/PEG 400/water (20:30:50, by vol.). At first, we performed in vitro studies with human oesophageal carcinoma cells KYSE 510, which were exposed to naked ORMOSIL NP, empty or loaded not covalently with mTHPC. The dark cytotoxicity of mTHPC was diminished by the entrapment in naked NP. The mTHPC delivery in naked NP reduced the cellular uptake of PS by about 50% in comparison to standard solvent, while surprisingly it did not affect the efficiency of cell photokilling. In fact, the dose-response curves for the phototoxicity (0.12 J/cm2 of red light) of free and NP-entrapped mTHPC were perfectly superimposed. Furthermore, mTHPC delivered by both formulations was rapidly internalised by KYSE 510 cells and localized preferentially in the Golgi apparatus and endoplasmic reticulum. Because of the very similar results obtained by delivering mTHPC by naked NP or standard solvent, we investigated the possible release of the PS from NP by two methods: a) FRET (Fluorescence Resonance Energy Transfer) experiments performed with NP loaded not covalently with mTHPC (donor) and a cyanine derivative (acceptor) covalently bound to the NP silanic matrix, and b) ultracentrifugation experiment of the NP dissolved in different solvents. Solvents of polarity lower than water, as well as the presence of serum proteins, caused the rapid and almost complete release of mTHPC from naked NPs. Studies on the monomerisation kinetics of mTHPC in the cell culture medium containing serum demonstrated an aggregation of mTHPC dissolved in standard vehicle, but not in NPs, that might explain the very similar efficiency of cell photoinactivation in spite of the lower cellular uptake of mTHPC delivered in NP with respect to the standard solvent. One strategy to avoid the leaking of the mTHPC from NP could be the coating of the NP surface with a suitable layer of PEG. These “stealthy particles” are characterized by a reduced protein adsorption on the surface, a long blood circulation time and minimum capture by the macrophages of the reticulo-endothelial system. The PEGylation of the ORMOSIL NPs strongly reduces, but not inhibit completely, the release of the physically entrapped mTHPC. For this reason PEGylated ORMOSIL NP loaded with mTHPC covalently bound to the silanic matrix were prepared. The dark cytotoxicity, intracellular uptake and distribution of PEGylated NP, empty and physically or covalently loaded with mTHPC, were determined in vitro in A549 cells, derived from human lung carcinoma, and normal human lung fibroblasts CCD-34Lu. The A549 cell line was selected because it is negative for the over-expression of folate receptor, and will be used in future experiments with nanovehicles functionalized with folic acid as ligand for specific targeting of tumour cells, together with KB cell line, which over-expresses the folate receptor. In both cell lines, the general lower dark and phototoxicity of mTHPC delivered by PEGylated NP, in comparison to the standard solvent, is very likely explained with the lower uptake of the drug. In cancer cells, but not in normal fibroblasts, reduction of viability caused by the exposure to increasing doses of mTHPC entrapped in PEGylated NP was partially due to the cytotoxicity of the empty PEGylated nanovehicle. This different response could depend from the presence in CCD-34Lu cells of efficient protection mechanisms against oxidative stress, as suggested from the high endogenous production of ROS in this cell line. In this project various types of unilamellar PEGylated liposomal formulations (Fospeg) were also studied as nanocarriers of mTHPC, which can be encapsulated in the phospholipidic bilayer. The liposomes used in this work were different for density (2-8 mol% of total lipids) and length (750, 2000, 5000) of PEG chains. We have used A549 and CCD-34Lu cells to evaluate the dark toxicity of mTHPC delivered by Fospeg in comparison to the delivery by standard solvent. We studied also how PEG chain density and length affect cellular uptake and PDT efficacy in cells in vitro. The encapsulation of mTHPC in PEGylated liposomes reduced drug cytotoxicity in the dark in both cell lines. The photosensitization efficiency of mTHPC toward the tumour cells in vitro was slightly reduced with the delivery by Fospeg in comparison to standard solvent and this correlated with the slightly lower uptake. Fluorescence microscopy revealed a rapid internalization of mTHPC and a cytoplasmic localization, mainly in the Golgi apparatus and endoplasmic reticulum, in both cell lines and irrespective of the modality of delivery (Fospeg or standard vehicle). To demonstrate if the PS was released from liposomes before the cellular internalization, we used the PEGylated liposomes labeled with a fluorescent lipid. A lysosomal localization was observed for liposomal probe Rhodamin-lipid suggesting that mTHPC was released from liposomes.
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La terapia fotodinamica (PDT) è una modalità terapeutica minimamente invasiva utilizzata prevalentemente in campo oncologico per il trattamento di vari tipi di tumori solidi. La PDT è basata sulla somministrazione di un agente fotosensibilizzante (PS) in grado di localizzarsi preferenzialmente nei tessuti neoplastici e di generare specie altamente reattive dell’ossigeno (ROS) dopo attivazione con luce visibile di opportune lunghezze d’onda. Tali specie, in particolare l’ossigeno di singoletto (1O2), innescano una cascata di reazioni portando a danno ossidativo e morte cellulare. Una limitazione della PDT è dovuta alla natura idrofobica di molti PS che, favorendo la sua aggregazione nel mezzo acquoso, ne diminuisce fortemente l’efficacia terapeutica. Per superare tale problema e migliorare l’efficacia e la selettività della PDT, limitando gli effetti citotossici nei tessuti sani circostanti, si può trarre vantaggio dalle potenzialità fornite dalle emergenti nanotecnologie. In oncologia, infatti, le nanostrutture stanno ricevendo particolare attenzione come nanovettori iniettabili per la veicolazione selettiva di farmaci alle cellule tumorali e come agenti per l’imaging e la diagnostica delle neoplasie. Il mio progetto di Dottorato di Ricerca è stato mirato sull’utilizzo di nanosistemi idrofilici per la veicolazione della meta-tetra(idrossifenil)clorina (mTHPC, nome commerciale Foscan®, fornita dalla Biolitec), un PS idrofobico di seconda generazione con un’elevata attività antitumorale ed approvato in Europa per il trattamento palliativo di tumori della testa e del collo in stadio avanzato. In particolare, nanoparticelle di silice organicamente modificata (ORMOSIL NP, ORganically MODified SILica nanoparticles; sintetizzate presso il Dipartimento di Scienze Chimiche, Università di Padova) e liposomi rivestiti di poli(etilene glicole) (PEG) (Fospeg; forniti dalla Biolitec) sono stati studiati come sistemi di veicolazione della mTHPC. L’incorporazione della mTHPC in NP non modifica le sue proprietà fotofisiche e la sua efficienza di produzione dell’1O2 dopo irradiamento. Sono stati valutati gli effetti biologici indotti da questi nanoveicoli in cellule umane tumorali e normali confrontando la citotossicità al buio, l’efficacia fotodinamica, l’internalizzazione e la distribuzione subcellulare della mTHPC incorporata in NP rispetto alla mTHPC veicolata nel solvente standard, costituito da etanolo/PEG 400/acqua (20:30:50, v/v/v). In primo luogo, sono stati condotti studi in vitro con cellule di carcinoma esofageo umano KYSE 510, che sono state esposte a ORMOSIL NP nude, non caricate e caricate non covalentemente con la mTHPC. La citotossicità indotta al buio dalla mTHPC è stata diminuita dall’incorporazione in NP nude. La veicolazione della mTHPC in NP nude riduce l’accumulo cellulare del PS di circa il 50% rispetto al veicolo standard, ma ciò inaspettatamente non va ad influire sul grado di foto citotossicità. Infatti, le curve dose-risposta relative alla fototossicità (0.12 J/cm2 di luce rossa) della mTHPC nella forma libera e incorporata in NP sono perfettamente sovrapponibili. Inoltre, la mTHPC veicolata in entrambe le formulazioni viene internalizzata rapidamente dalle cellule KYSE 510 e si localizza preferenzialmente nell’apparato del Golgi e nel reticolo endoplasmatico.
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