This thesis aims to develop and validate in vitro tests related to permeability, barrier effect and mucoadhesion to establish effectiveness and safety of nutraceuticals-like formulations. In vitro methodologies for permeability and barrier effect contemplate a multiparametric approach which includes the measurement of: i) the transmembrane passage of markers by using a static diffusion cells apparatus (Franz Cells) over a biomimetic tissue and, at the end of the experiment, ii) the evaluation of the membrane’s integrity (colorimetric test). In more details, the permeability assay, was firstly developed using model drugs (such as caffeine and acyclovir) and then validated by referring to literature data. Afterwards, the test was extended to nutraceutical products as well. With regards the tests on nutraceutical products, being the products mixture of ingredients, the permeability was determined by following defined active substances. The barrier effect was developed with a similar permeability-like approach by determining the caffeine passage across a biomimetic membrane treated with the product in exam. The results obtained was then compared with untreated membrane (negative control, without film-forming product) and membrane treated with Vaseline (positive control), a substance with film-forming action. In this case, caffeine was selected as a probe to assess the propensity of a given product to form a protective film due to its known ability to permeate different human tissues. Once completed permeability and barrier effect experiments, the integrity of the membrane was confirmed by colorimetric assay (methylene blue). The artificial biomimetic membranes, used in the hereabove described methods, consist of polymeric supports with different degree of porosity engineered with a mixture containing different concentrations of phospholipids, cholesterol and n-octanol, based on the compartment that they simulate. With this regard, during the thesis different biomimetic membranes will be produced and tested for simulating the gastro-intestinal, buccal, vaginal and skin tissues. Ex-vivo (porcine) sublingual tissue was used as well for testing the permeability of solid form nutraceutical products with patented delivery system. Lastly, product’s mucoadhesion and its resistance to body clearance mechanisms were evaluated through an inclined plane apparatus using a support functionalized with porcine gastric mucin. This dynamic test measures the retention of the mucoadhesive material on mucosal substrate (mucin film) and furthermore evaluates the interaction’s strength by simulating a physiological clearance mechanism reproduced by washing the surface with physiologic solution. In more details, mucoadhesion is calculated as difference in percentage between the amount of formulation loaded over the mucin and the formulation lost after a defined time and angle of inclination. The resistance against natural mechanisms of clearance is represented by the percentage of product which remains attached to the surface after the washing step. All data are compared with blank measurements performed in the absence of the mucin film. During the thesis work, the above-described in vitro tests were employed to study the performance of four different nutraceutical and topical formulations (i.e., oral gel against GERD, vaginal cream, cough syrup and solid formulations for the delivery of nutraceutical active components) in order to support the product development process and to define the correct product claims and classification in accordance with current European Regulations.
Questo studio di tesi mira a sviluppare e validare test in vitro di permeabilità, effetto barriera e mucoadesione per valutare l'efficacia e la sicurezza di formulazioni nutraceutiche. In dettaglio, i test in vitro di permeabilità ed effetto barriera si basano su un approccio multi-parametrico che include la misurazione di: i) il passaggio transmembrana di marker utilizzando la cella di Franz e un tessuto biomimetico e, alla fine dell'esperimento, ii) la valutazione dell'integrità della membrana (test colorimetrico). In una prima fase, per sviluppare il test di permeabilità, sono stati utilizzati farmaci modello (quali caffeina e aciclovir) e i dati ottenuti sono stati confrontati con riferimenti di letteratura al fine di validare il protocollo sperimentale sviluppato. Il test così ottimizzato è stato poi impiegato per valutare l’assorbimento di prodotti nutraceutici. Per questi ultimi, essendo costituiti da miscele complesse di ingredienti (attivi, eccipienti, etc.), viene determinata la permeabilità dei componenti attivi del prodotto. In una seconda fase di progetto, è stato sviluppato il test in vitro di effetto barriera per valutare la propensione di un dato prodotto a formare un film protettivo. In questo caso viene studiata la permeabilità della caffeina attraverso una membrana biomimetica precedentemente trattata con il prodotto in esame. I valori ottenuti sono poi comparati con il medesimo test di permeabilità della caffeina attraverso una membrana non trattata (controllo negativo) e una membrana trattata con vaselina (controllo positivo), sostanza dalle note proprietà protettive/filmogene. La caffeina è stata selezionata come marker vista la sua nota capacità di permeare diversi modelli di tessuti umani anche in assenza di danni. In tutti i casi sopra descritti, al termine dei test di permeabilità, l'integrità della membrana è stata confermata da un saggio colorimetrico (blu di metilene). Le membrane biomimetiche artificiali utilizzate durante i test sono composte da supporti polimerici, con diversi gradi di porosità, funzionalizzati, prima dell’utilizzo, con opportune miscele di fosfolipidi, colesterolo e n-ottanolo, al fine di mimare il comportamento delle membrane biologiche. A questo proposito, durante la tesi sono state prodotte e testate diverse membrane biomimetiche per mimare i tessuti gastro-intestinali, buccali, vaginali e cutanei. I test di mucoadesione, sviluppati con il metodo del piano inclinato impiegando superfici funzionalizzate con mucina gastrica suina, quantificano l’affinità con la mucina di una formulazione nutraceutica, nonché sua capacità di resistere ai meccanismi di clearance. Questo test dinamico misura l’adesione del prodotto nutraceutico sul substrato mucoso (film di mucina) e valuta inoltre la forza di interazione simulando il meccanismo di clearance corporeo grazie al lavaggio della formulazione con soluzione fisiologica (pH7.4). La mucoadesione viene calcolata come differenza in percentuale tra la quantità di formulazione caricata sulla mucina e la formulazione persa in un determinato tempo e con uno specifico angolo di inclinazione. La resistenza ai meccanismi naturali di clearance viene rappresentata come percentuale di prodotto che rimane attaccata alla superficie dopo la fase di lavaggio. Tutti i dati vengono confrontati con il bianco, eseguito in assenza del film di mucina. Nel corso della tesi, i test in vitro sopra descritti sono stati utilizzati per studiare quattro diversi prodotti nutraceutici e topici (gel orale per la GERD, crema vaginale e sciroppo per la tosse) al fine di valutarne le prestazioni, supportare il processo di sviluppo del prodotto e, infine, di definire il corretto claim e classificare il prodotto rispettando le correnti Normative Europee.
Sviluppo di test in vitro per valutare sicurezza ed efficacia di formulazioni nutraceutiche
BASSETTO, REBECCA
2021/2022
Abstract
This thesis aims to develop and validate in vitro tests related to permeability, barrier effect and mucoadhesion to establish effectiveness and safety of nutraceuticals-like formulations. In vitro methodologies for permeability and barrier effect contemplate a multiparametric approach which includes the measurement of: i) the transmembrane passage of markers by using a static diffusion cells apparatus (Franz Cells) over a biomimetic tissue and, at the end of the experiment, ii) the evaluation of the membrane’s integrity (colorimetric test). In more details, the permeability assay, was firstly developed using model drugs (such as caffeine and acyclovir) and then validated by referring to literature data. Afterwards, the test was extended to nutraceutical products as well. With regards the tests on nutraceutical products, being the products mixture of ingredients, the permeability was determined by following defined active substances. The barrier effect was developed with a similar permeability-like approach by determining the caffeine passage across a biomimetic membrane treated with the product in exam. The results obtained was then compared with untreated membrane (negative control, without film-forming product) and membrane treated with Vaseline (positive control), a substance with film-forming action. In this case, caffeine was selected as a probe to assess the propensity of a given product to form a protective film due to its known ability to permeate different human tissues. Once completed permeability and barrier effect experiments, the integrity of the membrane was confirmed by colorimetric assay (methylene blue). The artificial biomimetic membranes, used in the hereabove described methods, consist of polymeric supports with different degree of porosity engineered with a mixture containing different concentrations of phospholipids, cholesterol and n-octanol, based on the compartment that they simulate. With this regard, during the thesis different biomimetic membranes will be produced and tested for simulating the gastro-intestinal, buccal, vaginal and skin tissues. Ex-vivo (porcine) sublingual tissue was used as well for testing the permeability of solid form nutraceutical products with patented delivery system. Lastly, product’s mucoadhesion and its resistance to body clearance mechanisms were evaluated through an inclined plane apparatus using a support functionalized with porcine gastric mucin. This dynamic test measures the retention of the mucoadhesive material on mucosal substrate (mucin film) and furthermore evaluates the interaction’s strength by simulating a physiological clearance mechanism reproduced by washing the surface with physiologic solution. In more details, mucoadhesion is calculated as difference in percentage between the amount of formulation loaded over the mucin and the formulation lost after a defined time and angle of inclination. The resistance against natural mechanisms of clearance is represented by the percentage of product which remains attached to the surface after the washing step. All data are compared with blank measurements performed in the absence of the mucin film. During the thesis work, the above-described in vitro tests were employed to study the performance of four different nutraceutical and topical formulations (i.e., oral gel against GERD, vaginal cream, cough syrup and solid formulations for the delivery of nutraceutical active components) in order to support the product development process and to define the correct product claims and classification in accordance with current European Regulations.The text of this website © Università degli studi di Padova. Full Text are published under a non-exclusive license. Metadata are under a CC0 License
https://hdl.handle.net/20.500.12608/30964