Breast cancer (BC) is the most common cancer among women worldwide. In recent year nanotechnology has become of great interest in the biomedical field, and gold nanoparticles (AuNPs) are emerging as elective candidates for selective damaging of breast cancer. Medical application of these particular nanoparticles (NPs) require understanding of their interaction with biological system in order to control their physiological response, such as cellular uptake and cytotoxicity. AuNPs when are exposed to a biological fluids absorbed extracellular proteins on surface and formed the so-called protein corona (PC). The purpose of this work is to evaluate how the PC formation can affect gold nanoparticles behavior and how this interaction can mediate cells uptake and viability under in vitro cell culture conditions. In this thesis it was explored the formation of the PC after 1h, 3h and 16h of exposure of two different AuNPs shape (sphere or star) to cell culture media containing 10% of serum. In particular, three different culture media of three different cell lines were employed: NIH-3T3, a murine fibroblasts cell line used as healthy control; SKBR-3, cancer cell line overexpressing HER2 receptors and MCF-7, cancer cell line expressing ER-α receptors. The study was articulated in two main phases: in the first we focused on the formation of the protein corona. The PC formation was determined analyzing by UV-vis spectroscopy, DLS, electrophoresis and BCA. The results show the red-shift of the surface plasmon resonance peak, change of surface charge and the increase of the hydrodynamic diameter that indicated the conjugation of proteins to NPs, which was confirmed with BCA and SDS-Page too. In the second phase, to investigate how PC can affect AuNPs interaction with cells, four types conditions were set up: Condition 1: cells incubated in complete medium with AuNPs pre-incubated Condition 2: cells incubated in complete medium with AuNPs Condition 3: cells incubated in serum-free medium with AuNPs pre-incubated Condition 4: cells incubated in serum-free medium with AuNPs After incubation, cell viability was assessed using a cell viability assay, the MTT assay. The results show that the condition 1 and 3 demonstrated the most tolerability of AuNPs by cells, in particular it was highlighted by cell viability with AuNPs pre-incubated in serum-free medium. The reduction of cell viability in serum-free medium was around 50% with AuNPs not pre-incubated and around 10-20% in conditions 3. Condition 3 showed that the pre-incubation of gold nanoparticles led to a less reduction of cell viability compared to the AuNPs without PC. In all the conditions tested emerged tolerance by the healthy cells NIH-3T3, proving a specific effect towards cancer cells lines. At the light of these results, the experiments were focused on the serum-free medium condition. Uptaking studies by ICP-MS was performed. The results showed less uptake of pre-incubated AuNPs instead of the pristine AuNPs, which explains the results obtained from MTT assay. Furthermore, cellular internalization of the nanoparticles was confirmed by confocal laser scanning microscope. Despite several studies are still needed, overall from these preliminary data emerged the importance to keep in consideration the effect of protein corona with NPs in biological systems.
Il cancro al seno (BC) è il cancro più comune tra le donne in tutto il mondo. Negli ultimi anni la nanotecnologia è diventata di grande interesse nel campo biomedico, e le nanoparticelle d’oro (AuNPs) stanno emergendo come candidati elettivi per il danno selettivo del cancro al seno. L’applicazione medica di queste nanoparticelle particolari (NPs) richiede la comprensione della loro interazione con il sistema biologico al fine di controllare la loro risposta fisiologica, quali l’assorbimento cellulare e la citotossicità. AuNPs quando esposte ad un fluido biologico assorbono proteine extracellulari sulla superficie e formano la cosiddetta corona proteica (PC). Lo scopo di questo lavoro è di valutare come la formazione della PC può pregiudicare il comportamento delle nanoparticelle dell’oro e come questa interazione può mediare l’assorbimento delle cellule e la vitalità nelle circostanze della coltura cellulare in vitro. In questa tesi è stata studiata la formazione del PC dopo 1h, 3h e 16h di esposizione di due diverse forme di AuNPs (sfera o stella) a terreni di coltura cellulare contenenti il 10% di siero. In particolare, sono stati utilizzati tre diversi mezzi di coltura di tre diverse linee cellulari: NIH-3T3, una linea cellulare di fibroblasti murini utilizzata come controllo sano; SKBR-3, linea cellulare tumorale che sovraesprime i recettori HER2 e MCF-7, linea cellulare tumorale che esprime i recettori ER-α. Lo studio si è articolato in due fasi principali: nella prima ci siamo concentrati sulla formazione della corona proteica. La formazione del PC è stata determinata analizzando la spettroscopia UV-vis, DLS, elettroforesi e BCA. I risultati mostrano il red-shift del picco della banda plasmonica, il cambiamento della carica superficiale e l’aumento del diametro idrodinamico che indica la coniugazione delle proteine alle NPs, confermata anche con il BCA e SDS-Page. Nella seconda fase, per studiare come il PC può pregiudicare l’interazione di AuNPs con le celle, quattro tipi di condizioni sono state allestite: Condizione 1: cellule incubate in terreno completo con AuNP preincubate Condizione 2: cellule incubate in terreno completo con AuNPs Condizione 3: cellule incubate in terreno privo di siero con AuNP preincubate Condizione 4: cellule incubate in un terreno privo di siero con AuNPs Dopo l’incubazione, la vitalità cellulare è stata valutata utilizzando un test di vitalità cellulare, il test MTT. I risultati mostrano che le condizioni 1 e 3 hanno dimostrato la maggiore tollerabilità delle AuNPs da parte delle cellule, in particolare è stata evidenziata dalla vitalità cellulare con AuNPs pre-incubate in terreno privo di siero. La riduzione della vitalità cellulare nel terreno senza siero è stata di circa il 50% con le AuNPs non preincubate e di circa il 10-20% nella condizione 3. La condizione 3 ha mostrato che la pre-incubazione di nanoparticelle d’oro ha portato ad una minore riduzione della vitalità cellulare rispetto alle AuNPs senza PC. In tutte le condizioni testate è emersa la tolleranza da parte delle cellule sane NIH-3T3, dimostrando un effetto specifico verso le linee cellulari tumorali. Alla luce di questi risultati, gli esperimenti si sono concentrati sulla condizione di terreno senza siero. Sono stati condotti studi di uptake con ICP-MS. I risultati hanno mostrato un minore assorbimento delle AuNP pre-incubati rispetto alle AuNP non preincubate, il che spiega i risultati ottenuti dal test MTT. Inoltre, l’internalizzazione cellulare delle nanoparticelle è stata confermata dal microscopio confocale a scansione laser. Nonostante siano ancora necessari diversi studi, complessivamente da questi dati preliminari è emersa l’importanza di tenere conto dell’effetto della corona proteica nelle NPs nei sistemi biologici.
Valutazione in vitro di proteine corona-nanoparticelle d'oro sulla vitalità di cellule di carcinoma mammario
STRADA, SILVIA
2020/2021
Abstract
Breast cancer (BC) is the most common cancer among women worldwide. In recent year nanotechnology has become of great interest in the biomedical field, and gold nanoparticles (AuNPs) are emerging as elective candidates for selective damaging of breast cancer. Medical application of these particular nanoparticles (NPs) require understanding of their interaction with biological system in order to control their physiological response, such as cellular uptake and cytotoxicity. AuNPs when are exposed to a biological fluids absorbed extracellular proteins on surface and formed the so-called protein corona (PC). The purpose of this work is to evaluate how the PC formation can affect gold nanoparticles behavior and how this interaction can mediate cells uptake and viability under in vitro cell culture conditions. In this thesis it was explored the formation of the PC after 1h, 3h and 16h of exposure of two different AuNPs shape (sphere or star) to cell culture media containing 10% of serum. In particular, three different culture media of three different cell lines were employed: NIH-3T3, a murine fibroblasts cell line used as healthy control; SKBR-3, cancer cell line overexpressing HER2 receptors and MCF-7, cancer cell line expressing ER-α receptors. The study was articulated in two main phases: in the first we focused on the formation of the protein corona. The PC formation was determined analyzing by UV-vis spectroscopy, DLS, electrophoresis and BCA. The results show the red-shift of the surface plasmon resonance peak, change of surface charge and the increase of the hydrodynamic diameter that indicated the conjugation of proteins to NPs, which was confirmed with BCA and SDS-Page too. In the second phase, to investigate how PC can affect AuNPs interaction with cells, four types conditions were set up: Condition 1: cells incubated in complete medium with AuNPs pre-incubated Condition 2: cells incubated in complete medium with AuNPs Condition 3: cells incubated in serum-free medium with AuNPs pre-incubated Condition 4: cells incubated in serum-free medium with AuNPs After incubation, cell viability was assessed using a cell viability assay, the MTT assay. The results show that the condition 1 and 3 demonstrated the most tolerability of AuNPs by cells, in particular it was highlighted by cell viability with AuNPs pre-incubated in serum-free medium. The reduction of cell viability in serum-free medium was around 50% with AuNPs not pre-incubated and around 10-20% in conditions 3. Condition 3 showed that the pre-incubation of gold nanoparticles led to a less reduction of cell viability compared to the AuNPs without PC. In all the conditions tested emerged tolerance by the healthy cells NIH-3T3, proving a specific effect towards cancer cells lines. At the light of these results, the experiments were focused on the serum-free medium condition. Uptaking studies by ICP-MS was performed. The results showed less uptake of pre-incubated AuNPs instead of the pristine AuNPs, which explains the results obtained from MTT assay. Furthermore, cellular internalization of the nanoparticles was confirmed by confocal laser scanning microscope. Despite several studies are still needed, overall from these preliminary data emerged the importance to keep in consideration the effect of protein corona with NPs in biological systems.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14585