Traditional anticancer chemotherapy involves the systemic use of cytotoxic drugs which are distributed in a non-specific way through the entire body and can often induce severe side effects. In this context, nanostructured systems for drug delivery may be used for site-specific targeting of anticancer drugs, improving therapeutic efficiency and reducing side effects. Silk fibroin, a protein produced by Bombyx mori, emerges among the natural polymers for the production of nanoparticles, due to its excellent biocompatibility, biodegradability, ability to self-organize and easy functionalization. Silk fibroin nanoparticles (SFN), normally directed to the tumor by passive targeting, can be functionalized on their surface with specific ligands which, recognizing certain receptors / antigens present on the target cells, endow them with active targeting. In this regard, integrins can play a key role in tumor targeting due to their overexpression by many types of cancer cells; the tripeptide motif Arg-Gly-Asp (RGD) is known for being recognized by members of the integrin superfamily and present itself as an excellent candidate for functionalization to achieve targeted targeting. In this thesis work, SFNs were prepared, loaded with curcumin and functionalized with cRGD to provide them with an active targeting property towards tumor cells. Curcumin was chosen as a model molecule for poorly soluble and poorly bioavailable drugs. SFNs were prepared using the acetone desolvation method in which curcumin powder was first solubilized; subsequently they were functionalized with cRGD, with high affinity for the αvβ3 integrin which is highly expressed in the endothelial cells of the tumor vascular system or in tumor cells, through a “click reaction”. Results showed that naked SFNs have an average diameter of 128,9± 2,4 nm and functionalization increased dimensions up to an average diameter of 142,7 ± 2,7 nm. Cellular metabolic activity was tested on different cell lines i) mesenchymal stem cells (AD-MSC), ii) human fibroblast (HF), iii) Caucasian colorectal adenocarcinoma cells (Caco-2), iv) cancer cells of human cervix (HeLa), and v) human urinary bladder cancer cells (ECV). Cells incubated with naked SFNs remained nearly 100% viable compared to control (SFNs concentration = 0) for all tested doses, while cRGD-functionalized SFNs showed a more cytotoxic effect on cell lines. cRGD-functionalized SFNs are internalized by tumor cells that overexpress the integrin receptors (but not by cells that do not overexpress them), especially at the lowest concentration tested (0.01 mg / ml). On the contrary, at higher concentrations, non-specific interactions between SFNs and cell membrane are promoted and coupled to specific absorption. Cellular uptake of SFNs was then confirmed by microscopy, exploiting fibroin and curcumin fluorescence, using Caco-2 which do not overexpress integrin receptors, and ECV, which instead overexpress them. For Caco-2 cells, no different behavior was detected between functionalized and naked SFNs, and a slight internalization of functionalized SFNs was observed only after 6 hours; naked SFNs were internalized by ECVs only after 6 hours of incubation, while a marked absorption of functionalized SFNs was recorded already after 1 hour of incubation. In conclusion, SFNs loaded with curcumin and functionalized with cRGD provided an in vitro active targeting for the site-specific release of anticancer drug, enhancing the activity and sparing healthy cells.
La chemioterapia antitumorale tradizionale prevede l’impiego per via sistemica di farmaci citotossici che si distribuiscono in modo non specifico in tutto l’organismo e inducono effetti collaterali più o meno gravi. In questo contesto, i sistemi nanostrutturati per il drug delivery possono essere utilizzati per il direzionamento sito-specifico di tali farmaci migliorandone l’efficienza terapeutica e riducendo gli effetti collaterali. La fibroina, proteina prodotta dal Bombyx mori, emerge tra i polimeri naturali per la produzione dei sistemi nanoparticellari grazie alla sua eccellente biocompatibilità, biodegradabilità, capacità di auto-organizzazione e facile funzionalizzazione. Le nanoparticelle di fibroina di seta (SFN), normalmente direzionate al tumore mediante targeting passivo, possono essere funzionalizzate sulla superficie con ligandi specifici che, riconoscendo determinati recettori/antigeni presenti sulle cellule bersaglio, le dotano di un targeting attivo. A tal proposito le integrine possono svolgere un ruolo fondamentale nel targeting tumorale a causa della loro sovraespressione da parte di molti tipi di cellule tumorali; il tripeptide Arg-Gly-Asp (RGD) è riconosciuto dai membri della superfamiglia delle integrine e si presenta come un ottimo candidato per la funzionalizzazione e l’ottenimento di un targeting mirato. In questo lavoro di tesi, sono state preparate SFN caricate di curcumina e funzionalizzate con cRGD per dotarle di proprietà di targeting attivo verso le cellule tumorali. Le SFN sono state preparate tramite il metodo di desolvatazione in acetone nel quale è stata prima solubilizzata la polvere di curcumina; poi sono state funzionalizzate con cRGD, con elevata affinità per l’integrina αvβ3, altamente espressa nelle cellule endoteliali del sistema vascolare del tumore o nelle cellule tumorali, tramite una “click reaction”. I risultati hanno mostrato che le SFN non funzionalizzate hanno diametro medio di 128,9 ± 2,4 nm e che la funzionalizzazione ha aumentato le dimensioni ad un diametro medio di 142,7 ± 2,7 nm. L’attività metabolica cellulare è stata testata su diverse linee cellulari i) cellule staminali mesenchimali, ii) fibroblasti umani, iii) cellule di adenocarcinoma colorettale caucasico (Caco-2), iv) cellule di cancro della cervice umana e v) cellule tumorali della vescica urinaria umana (ECV). Le cellule incubate con le SFN non funzionalizzate sono rimaste vitali quasi al 100% rispetto al controllo (concentrazione di SFN=0) per tutte le dosi testate, mentre i nanosistemi funzionalizzati con cRGD hanno mostrato un maggior effetto citotossico sulle linee cellulari. Le SFN funzionalizzate con cRGD vengono internalizzate dalle cellule tumorali che sovraesprimono i recettori delle integrine (ma non dalle cellule che non li sovraesprimono), soprattutto alla concentrazione più bassa testata (0,01 mg/ml). Al contrario, a concentrazioni più elevate, le interazioni aspecifiche tra SFN e membrana cellulare sono promosse e accoppiate all’assorbimento specifico. L’uptake cellulare delle SFN è stato poi confermato mediante microscopia, sfruttando la fluorescenza della fibroina e della curcumina, utilizzando le Caco-2, che non sovraesprimono i recettori dell’integrina, e le ECV, che invece li sovraesprimono. Per le cellule Caco-2 non è stato rilevato un diverso comportamento tra le SFN funzionalizzate e quelle non, ed è stata osservata una leggera internalizzazione delle SFN funzionalizzate solo dopo 6 ore; le SFN non funzionalizzate sono state internalizzate dalle ECV solo dopo 6 ore di incubazione, mentre è stato registrato un marcato assorbimento delle SFN funzionalizzate già dopo 1 ora di incubazione. In conclusione, la funzionalizzazione con cRGD delle SFN caricate con curcumina ha fornito un targeting attivo in vitro per la liberazione sito-specifica del farmaco.
Targeting tumorale attivo tramite l'utilizzo di nanoparticelle di fibroina funzionalizzate con cRGD
COSTANZO, NICOLE
2019/2020
Abstract
Traditional anticancer chemotherapy involves the systemic use of cytotoxic drugs which are distributed in a non-specific way through the entire body and can often induce severe side effects. In this context, nanostructured systems for drug delivery may be used for site-specific targeting of anticancer drugs, improving therapeutic efficiency and reducing side effects. Silk fibroin, a protein produced by Bombyx mori, emerges among the natural polymers for the production of nanoparticles, due to its excellent biocompatibility, biodegradability, ability to self-organize and easy functionalization. Silk fibroin nanoparticles (SFN), normally directed to the tumor by passive targeting, can be functionalized on their surface with specific ligands which, recognizing certain receptors / antigens present on the target cells, endow them with active targeting. In this regard, integrins can play a key role in tumor targeting due to their overexpression by many types of cancer cells; the tripeptide motif Arg-Gly-Asp (RGD) is known for being recognized by members of the integrin superfamily and present itself as an excellent candidate for functionalization to achieve targeted targeting. In this thesis work, SFNs were prepared, loaded with curcumin and functionalized with cRGD to provide them with an active targeting property towards tumor cells. Curcumin was chosen as a model molecule for poorly soluble and poorly bioavailable drugs. SFNs were prepared using the acetone desolvation method in which curcumin powder was first solubilized; subsequently they were functionalized with cRGD, with high affinity for the αvβ3 integrin which is highly expressed in the endothelial cells of the tumor vascular system or in tumor cells, through a “click reaction”. Results showed that naked SFNs have an average diameter of 128,9± 2,4 nm and functionalization increased dimensions up to an average diameter of 142,7 ± 2,7 nm. Cellular metabolic activity was tested on different cell lines i) mesenchymal stem cells (AD-MSC), ii) human fibroblast (HF), iii) Caucasian colorectal adenocarcinoma cells (Caco-2), iv) cancer cells of human cervix (HeLa), and v) human urinary bladder cancer cells (ECV). Cells incubated with naked SFNs remained nearly 100% viable compared to control (SFNs concentration = 0) for all tested doses, while cRGD-functionalized SFNs showed a more cytotoxic effect on cell lines. cRGD-functionalized SFNs are internalized by tumor cells that overexpress the integrin receptors (but not by cells that do not overexpress them), especially at the lowest concentration tested (0.01 mg / ml). On the contrary, at higher concentrations, non-specific interactions between SFNs and cell membrane are promoted and coupled to specific absorption. Cellular uptake of SFNs was then confirmed by microscopy, exploiting fibroin and curcumin fluorescence, using Caco-2 which do not overexpress integrin receptors, and ECV, which instead overexpress them. For Caco-2 cells, no different behavior was detected between functionalized and naked SFNs, and a slight internalization of functionalized SFNs was observed only after 6 hours; naked SFNs were internalized by ECVs only after 6 hours of incubation, while a marked absorption of functionalized SFNs was recorded already after 1 hour of incubation. In conclusion, SFNs loaded with curcumin and functionalized with cRGD provided an in vitro active targeting for the site-specific release of anticancer drug, enhancing the activity and sparing healthy cells.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/20.500.14239/12532