The silk produced by the silkworm (Bombyx mori) has an inner core consisting of two fibroin protein filaments, bound by a protein that acts as a glue, sericin. The process called “degumming” allows separation of fibroin and sericin solubilizing the latter through different agents and methods. Only recently the biologic properties of sericin have been exploited for biomedical and pharmaceutical purposes: this protein is little used for the formulation of nanoparticle systems for drug delivery, despite its excellent biocompatibility, controllable biodegradability and non-immunogenicity. The goal of this thesis was to develop sericin nanoparticles (SNP) that guaranteed the controlled release of natural polyphenols (epigallocatechin gallate, E; proanthocyanidins, P and quercetin, Q) in order to support tissue regeneration by resident mesenchymal stem cells (MSC). The cocoons of Bombyx mori have been cut into pieces and degummed with an autoclave at 120° C for 1 h; the obtained sericin solution has been filtered through 70 µm cell sieves, frozen at -80° C and finally freeze-dried. The freeze-dried sericin powder and Pluronic® F-127 excipient have been solubilized in DMSO with a final concentration respectively of 0.5 and 2.5% (w/v). The drug has been added at the solution at the concentration of 0.1 % (w/v) and kept under magnetic stirring at 37 °C until the complete desolvation. The obtained solution has been added drop by drop at the deionized water under magnetic stirring, permitting the development of self-assembling nanoparticles. The resulting suspension of nanoparticles has been later dialyzed, sonicated and centrifuged in order to remove aggregates, and freeze-dried. The morphological investigation performed by SEM confirmed the nanometric size of SNPs and their spherical shape with a smooth surface. Process yield (%) in SNP preparations ranged from 60.9 ± 0.46% for SNP-Q to 63.8 ± 4.25% for SNP-P. The nanoparticles have been able to encapsulate both hydrophilic (P and E) and hydrophobic (Q) drugs thanks to the formation of micelles. Regarding the drug loading and encapsulation efficiency, the statistical analysis did not reveal significant differences between different batches of the same drug, but between the different drugs considered. The encapsulation efficiency has resulted better for hydrophilic drugs. The drug release from SNPs has been studied evaluating two different dissolution media: EtOH and PBS. A slow and controlled release profile has been obtained for all drugs in PBS; in EtOH, a burst-effect has been observed for Q and E but not for P. Processing the data regarding drug release using kinetic models revealed that P has a lower diffusion index than the other biomolecules, which is reasonable given its greater steric hindrance. All the drugs have revealed excellent antioxidant, anti-elastase and anti-tyrosinase properties in vitro; after encapsulation, antioxidant activity has been preserved for Q and E and reduced for P. The anti-elastase activity was lower for SNP-P and SNP-E than the activity of the equivalent amount of free drug; the anti-tyrosinase activity of both P and Q decreased as a result of encapsulation in SNPs; only E, when encapsulated, retained good inhibitory properties. All the formulations have promoted the metabolic activity of MSCs for over 72 hours and have protected the cells from damage caused by oxidative stress. Overall, the results obtained, support the use of SNPs to address the natural origin flavonoids to MSCs resident in tissues, for regenerative purposes.
La seta prodotta dal baco da seta (Bombyx mori) ha un nucleo interno costituito da due filamenti proteici di fibroina, legati da una proteina che funge da collante, la sericina. Il processo denominato “sgommatura” permette la separazione di fibroina e sericina solubilizzando quest’ultima con diversi agenti e metodi. Solo recentemente le proprietà biologiche della sericina sono state sfruttate per scopi biomedici e farmaceutici: nel campo del drug delivery questa proteina è poco impiegata nella formulazione di sistemi nanoparticellari per la veicolazione di farmaci, nonostante la sua eccellente biocompatibilità, biodegradabilità controllabile e non immunogenicità. Lo scopo di questa tesi è stato quello di sviluppare nanoparticelle di sericina (SNP) che garantissero il rilascio controllato di polifenoli naturali (epigallocatechingallato, E; proantocianidine, P e quercetina, Q) per sostenere la rigenerazione tissutale da parte delle cellule staminali mesenchimali (MSC) residenti. I bozzoli di Bombyx mori sono stati tagliati a pezzi e sgommati in autoclave a 120° C per 1h; la soluzione di sericina ottenuta è stata filtrata mediante setacci cellulari da 70 µm, congelata a -80° C ed infine liofilizzata. La polvere di sericina liofilizzata e l’eccipiente Pluronic® F-127 sono stati solubilizzati in DMSO alla concentrazione finale rispettivamente di 0,5 e 2,5% (p/v). Il principio attivo è stato aggiunto alla soluzione in concentrazione pari allo 0,1% (p/v) e mantenuto in agitazione magnetica a 37° C fino a completa dissoluzione. La soluzione ottenuta è stata aggiunta goccia a goccia all’acqua deionizzata in agitazione magnetica, permettendo la formazione delle nanoparticelle per self-assembling. La sospensione di nanoparticelle ottenuta è stata in seguito dializzata, sonicata e centrifugata per eliminare gli aggregati, e liofilizzata. L’indagine morfologica eseguita mediante SEM ha confermato la dimensione nanometrica delle SNP e la loro forma sferica con superficie liscia. La resa del processo (%) nelle preparazioni di SNP variava dal 60,9 ± 0,46% per SNP-Q al 63,8 ± 4,25% per SNP-P. Le nanoparticelle sono state in grado di incapsulare sia farmaci idrofili (P ed E) che idrofobici (Q) grazie alla formazione di micelle. Per quanto riguarda il caricamento del farmaco e l’efficienza di incapsulamento, l’analisi statistica non ha rivelato differenze significative tra diversi lotti dello stesso principio attivo, bensì tra i diversi attivi considerati. L’efficienza di incapsulamento è risultata più elevata per i principi attivi idrofili. Il rilascio del farmaco dalle SNP è stato studiato considerando due mezzi di dissoluzione: EtOH e PBS. In PBS è stato ottenuto un profilo di rilascio lento e controllato per tutti gli attivi; in EtOH è stato riscontrato un burst-effect per Q ed E ma non per P. L’elaborazione dei dati riguardanti il release del farmaco tramite modelli cinetici ha rivelato che P presenta un indice di diffusione inferiore rispetto alle altre biomolecole, il che è ragionevole dato il suo maggior impedimento sterico. Tutti i principi attivi hanno mostrato eccellenti proprietà antiossidanti, antielastasiche ed antitirosinasiche in vitro; in seguito ad incapsulamento, l’attività antiossidante è stata preservata per Q ed E e ridotta per P. L’attività antielastasica è risultata inferiore per SNP-P e SNP-E rispetto all’attività della quantità equivalente di farmaco libero; l’attività antitirosinasica sia di P che di Q è diminuita in seguito all’incapsulamento nelle SNP; solo E, quando incapsulato, ha conservato buone proprietà inibitorie. Tutte le formulazioni hanno promosso l’attività metabolica delle MSC per oltre 72 ore e hanno protetto le cellule dal danno causato dallo stress ossidativo. Nel complesso, i risultati ottenuti, supportano l’impiego di SNP per indirizzare i flavonoidi di origine naturale alle MSC residenti nei tessuti, a fini rigenerativi.
Nanoparticelle di sericina a rilascio controllato di polifenoli per la rigenerazione dei tessuti mediata dalle cellule mesenchimali
LOCATELLI, GIULIA
2019/2020
Abstract
The silk produced by the silkworm (Bombyx mori) has an inner core consisting of two fibroin protein filaments, bound by a protein that acts as a glue, sericin. The process called “degumming” allows separation of fibroin and sericin solubilizing the latter through different agents and methods. Only recently the biologic properties of sericin have been exploited for biomedical and pharmaceutical purposes: this protein is little used for the formulation of nanoparticle systems for drug delivery, despite its excellent biocompatibility, controllable biodegradability and non-immunogenicity. The goal of this thesis was to develop sericin nanoparticles (SNP) that guaranteed the controlled release of natural polyphenols (epigallocatechin gallate, E; proanthocyanidins, P and quercetin, Q) in order to support tissue regeneration by resident mesenchymal stem cells (MSC). The cocoons of Bombyx mori have been cut into pieces and degummed with an autoclave at 120° C for 1 h; the obtained sericin solution has been filtered through 70 µm cell sieves, frozen at -80° C and finally freeze-dried. The freeze-dried sericin powder and Pluronic® F-127 excipient have been solubilized in DMSO with a final concentration respectively of 0.5 and 2.5% (w/v). The drug has been added at the solution at the concentration of 0.1 % (w/v) and kept under magnetic stirring at 37 °C until the complete desolvation. The obtained solution has been added drop by drop at the deionized water under magnetic stirring, permitting the development of self-assembling nanoparticles. The resulting suspension of nanoparticles has been later dialyzed, sonicated and centrifuged in order to remove aggregates, and freeze-dried. The morphological investigation performed by SEM confirmed the nanometric size of SNPs and their spherical shape with a smooth surface. Process yield (%) in SNP preparations ranged from 60.9 ± 0.46% for SNP-Q to 63.8 ± 4.25% for SNP-P. The nanoparticles have been able to encapsulate both hydrophilic (P and E) and hydrophobic (Q) drugs thanks to the formation of micelles. Regarding the drug loading and encapsulation efficiency, the statistical analysis did not reveal significant differences between different batches of the same drug, but between the different drugs considered. The encapsulation efficiency has resulted better for hydrophilic drugs. The drug release from SNPs has been studied evaluating two different dissolution media: EtOH and PBS. A slow and controlled release profile has been obtained for all drugs in PBS; in EtOH, a burst-effect has been observed for Q and E but not for P. Processing the data regarding drug release using kinetic models revealed that P has a lower diffusion index than the other biomolecules, which is reasonable given its greater steric hindrance. All the drugs have revealed excellent antioxidant, anti-elastase and anti-tyrosinase properties in vitro; after encapsulation, antioxidant activity has been preserved for Q and E and reduced for P. The anti-elastase activity was lower for SNP-P and SNP-E than the activity of the equivalent amount of free drug; the anti-tyrosinase activity of both P and Q decreased as a result of encapsulation in SNPs; only E, when encapsulated, retained good inhibitory properties. All the formulations have promoted the metabolic activity of MSCs for over 72 hours and have protected the cells from damage caused by oxidative stress. Overall, the results obtained, support the use of SNPs to address the natural origin flavonoids to MSCs resident in tissues, for regenerative purposes.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/20.500.14239/12116