A spectrofluorometric analysis to evaluate transcutaneous biodistribution of fluorescent nanoparticulate gel formulations

The investigation of the absorption of drug delivery systems, designed for the transport of therapeutic molecules inside the body, could be relatively simplified by the fluorophore association and tracking by means of bio-imaging techniques (i.e., optical in vivo imaging or confocal and multiphoton microscopy). However, when a fluorescence signal comes out from the skin, its specific detection can be problematic. Skin high autofluorescence can hinder the observation of administered exogenous fluorophores conjugated to drug delivery systems, making it more challenging to detect their biodistribution. In the present study, we have developed a method based on the spectrofluorometric analysis of skin samples to discriminate the fluorescent signal coming from administered fluorescent molecules from the background. Moreover, we gave a semi-quantitative evaluation of the signal intensity. Thus, we distinguished two gel formulations loading the fluorophore rhodamine B (called GEL RHO and GEL SLN-RHO). The two formulations of gels, one of which containing solid lipid nanoparticles (GEL RHO-SLN), were administered on skin explants incubated in a bioreactor, and the penetration was evaluated at different time points (2 and 6 hours). Cryostatic sections of skin samples were observed with confocal laser scanning microscopy, and a spectrofluorometric analysis was performed. Significantly higher signal intensity in the samples administered with SLN-RHO GEL, with a preferential accumulation in the hair bulbs, was found. Reaching also the deeper layers of the hair shaft after 6 hours, the solid lipid nanoparticles thickened with polymer represent a suitable drug delivery system for transcutaneous administration

Niosomes for Topical Application of Antioxidant Molecules: Design and In Vitro Behavior

In the present study, gels based on xanthan gum and poloxamer 407 have been developed and characterized in order to convey natural antioxidant molecules included in niosomes. Specifically, the studies were conducted to evaluate how the vesicular systems affect the release of the active ingredient and which formulation is most suitable for cutaneous application. Niosomes, composed of Span 20 or Tween 20, were produced through the direct hydration method, and therefore, borate buffer or a micellar solution of poloxamer 188 was used as the aqueous phase. The niosomes were firstly characterized in terms of morphology, dimensional and encapsulation stability. Afterwards, gels based on poloxamer 407 or xanthan gum were compared in terms of spreadability and adhesiveness. It was found to have greater spreadability for gels based on poloxamer 407 and 100% adhesiveness for those based on xanthan gum. The in vitro diffusion of drugs studied using Franz cells associated with membranes of mixed cellulose esters showed that the use of a poloxamer micellar hydration phase determined a lower release as well as the use of Span 20. The thickened niosomes ensured controlled diffusion of the antioxidant molecules. Lastly, the in vivo irritation test confirmed the safeness of niosomal gels after cutaneous application

Ellagic acid containing Nanostructured Lipid Carriers for topical application: a preliminary study

Ellagic acid (EA) is a potent antioxidant substance of natural origin characterized by poor biopharmaceutical properties and low solubility in water that limit their use. The aim of the present study was to develop lipid based nanoparticle formulations able to encapsulate EA for dermal delivery purpose. The EA-loaded nanoparticles were prepared using two different lipid compositions, namely tristearin/tricaprylin (NLC-EA1) and tristearin/labrasol (NLC-EA2). The influence of formulations on size, entrapment efficiency and stability of EA-loaded nanoparticles was investigated. Cryo-TEM and SAXS analysess showed that no morphological differences are evident among all the types of loaded and unloaded NLC. The macroscopic aspect of both NLC-EA1 and NLC-EA2 did not change by time. No difference in size is appreciable between empty and drug-containing NLC, thus the nanoparticle diameter is not affected by the presence EA and in general no variations of the diameters occur during time. The percentage of entrapment efficiency of both EA-loaded nanoparticles was almost quantitative. In addition NLC-EA1 maintain EA stability for almost 2 months, while NLC-EA2 up to 40 days. FRAP assay showed an antioxidant activity around 60% for both the loaded NLC, as compared to the solution. Although both types of NLC are characterized by some toxicity, NLC-EA1 are less cytotoxic than NLC-EA2. Taken together these results demonstrated that the inclusion of EA within NLC could improve the water solubility, allowing for a reduction of the dosage. Moreover, the maintaining of high antioxidant effect and low toxicity were evidenced for both types of NLC-EA

Formulative Study and Intracellular Fate Evaluation of Ethosomes and Transethosomes for Vitamin D3 Delivery

In this pilot study, ethosomes and transethosomes were investigated as potential delivery systems for cholecalciferol (vitamin D3), whose deficiency has been correlated to many disorders such as dermatological diseases, systemic infections, cancer and sarcopenia. A formulative study on the influence of pharmaceutically acceptable ionic and non-ionic surfactants allowed the preparation of different transethosomes. In vitro cytotoxicity was evaluated in different cell types representative of epithelial, connective and muscle tissue. Then, the selected nanocarriers were further investigated at light and transmission electron microscopy to evaluate their uptake and intracellular fate. Both ethosomes and transethosomes proven to have physicochemical properties optimal for transdermal penetration and efficient vitamin D3 loading; moreover, nanocarriers were easily internalized by all cell types, although they followed distinct intracellular fates: ethosomes persisted for long times inside the cytoplasm, without inducing subcellular alteration, while transethosomes underwent rapid degradation giving rise to an intracellular accumulation of lipids. These basic results provide a solid scientific background to in vivo investigations aimed at exploring the efficacy of vitamin D3 transdermal administration in different experimental and pathological conditions

Ethosomes and transethosomes for mangiferin transdermal delivery

Mangiferin is a natural glucosyl xanthone with antioxidant and anti-inflammatory activity, making it suitable for protection against cutaneous diseases. In this study ethosomes and transethosomes were designed as topical delivery systems for mangiferin. A preformulation study was conducted using different surfactants in association with phosphatidylcholine. Vesicle dimensional distribution was monitored by photon correlation spectroscopy, while antioxidant capacity and cytotoxicity were respectively assessed by free radical scavenging analysis and MTT on HaCaT keratinocytes. Selected nanosystems were further investigated by cryogenic transmission electron microscopy, while mangiferin entrapment capacity was evaluated by ultracentrifugation and HPLC. The diffusion kinetics of mangiferin from ethosomes and transethosomes evaluated by Franz cell was faster in the case of transethosomes. The suitability of mangiferin-containing nanovesicles in the treatment of skin disorders related to pollutants was investigated, evaluating, in vitro, the antioxidant and anti-inflammatory effect of ethosomes and transethosomes on human keratinocytes exposed to cigarette smoke as an oxidative and inflammatory challenger. The ability to induce an antioxidant response (HO-1) and anti-inflammatory status (IL-6 and NF-kB) was determined by RT-PCR and immunofluorescence. The data demonstrated the effectiveness of mangiferin loaded in nanosystems to protect cells from damage. Finally, to gain insight into the keratinocytes' uptake of ethosome and transethosome, transmission electron microscopy analyses were conducted, showing that both nanosystems were able to pass intact within the cells

Manganese in Diagnostics: A Preformulatory Study

This investigation aims to find lipid-based nanosystems to be used as tools to deliver manganese for diagnostic purposes in multimodal imaging techniques. In particular, the study describes the production and characterization of aqueous dispersions of anionic liposomes as delivery systems for two model manganese-based compounds, namely manganese chloride and manganese acetylacetonate. Negatively charged liposomes were obtained using four different anionic surfactants, namely sodium docusate (SD), N-lauroylsarcosine (NLS), Protelan AG8 (PAG) and sodium lauroyl lactylate (SLL). Liposomes were produced by the direct hydration method followed by extrusion and characterized in terms of size, polydispersity, surface charge and stability over time. After extrusion, liposomes are homogeneous and monodispersed with an average diameter not exceeding 200 nm and a negative surface charge as confirmed by ζ potential measurement. Moreover, as indicated by atomic absorption spectroscopy analyses, the loading of manganese-based compounds was almost quantitative. Liposomes containing NLS or SLL were the most stable over time and the presence of manganese-based compounds did not affect their size distribution. Liposomes containing PAG and SD were instable and therefore discarded. The in vitro cytotoxicity of the selected anionic liposomes was evaluated by MTT assay on human keratinocyte. The obtained results highlighted that the toxicity of the formulations is dose dependent

Nanotechnological strategies for topical administration of active compounds

La pelle è l'organo più esteso del corpo umano e rappresenta un sito importante per l'assorbimento di numerose molecole attive. La somministrazione topica dei farmaci risulta quindi essere una strategia efficace per il trattamento di molte patologie locali e sistemiche, presentando molti vantaggi rispetto alle tradizionali vie di somministrazione orale e parenterale. Le preparazioni topiche convenzionali presentano molti limiti nel rilascio del principio attivo nella circolazione sistemica, quindi sono stati sviluppati dei sistemi di veicolazione innovativi caratterizzati da dimensioni micro e nanometriche. I principali vantaggi di questi sistemi sono il rilascio mirato sul sito d’azione e l’aumento della stabilità del farmaco incapsulato. In particolare, i sistemi di rilascio lipidici, grazie alla loro biodegradabilità, bassa tossicità e somiglianza con i lipidi dell'epidermide risultano promettenti per l’applicazione topica. In questo progetto, Liposfere (LS), Nanoparticelle Solide Lipidiche (SLN), Vettori Lipidici Nanostrutturati (NLC), Etosomi (ETHO) e Gel di Poloxamer sono stati studiati come sistemi di veicolazione per la somministrazione topica di diverse molecole. In seguito alla loro caratterizzazione, sono stati descritti esperimenti in vitro tecnologici e biologici, ex vivo o in vivo per verificare l'efficacia delle formulazioni sviluppate. Liposfere contenenti Clotrimazolo, veicolate all’interno di un gel, sono state studiate per il trattamento di Candida albicans. La caratterizzazione dimensionale, morfologica, di efficienza di incapsulamento, viscosità e adesione, gli studi in vitro sull’attività antimicotica e di rilascio hanno permesso di validare la loro efficacia. È stato poi effettuato uno studio di scaling-up incentrato sulla produzione di nanoparticelle contenenti Progesterone. Sono stati effettuati studi dimensionali, morfologici, di efficienza dell'incapsulamento e di stabilità delle nanoparticelle prodotte in scala industriale. Inoltre, sono stati realizzati studi in vitro di rilascio e di permeazione cutanea rispettivamente con metodi di dialisi e celle di Franz, e studi in vivo mediante tape stripping. Gel nanoparticellari sono stati prodotti per veicolare Acido Ialuronico e Retinil Palmitato per la cicatrizzazione delle ferite, generando un sistema adatto alla somministrazione cutanea. Sono stati quindi eseguiti test di “wound healing” in vitro per studiare l’attività del principio attivo somministrato tramite gel nanoparticellare. SLN e NLC sono state sviluppate anche per il rilascio di -tocoferolo con l'obiettivo di proteggere la pelle umana da inquinanti, come il fumo di sigaretta. Uno studio preformulativo ha permesso di selezionare la composizione ottimale delle nanoparticelle e l'effetto antiossidante di -tocoferolo incapsulato è stato studiato attraverso esperimenti ex vivo su biopsie umane. NLC ed ETHO contenenti Ubichinone sono stati prodotti e confrontati con l'obiettivo di formulare una nuova strategia terapeutica per il trattamento della sindrome di Rett. La scarsa stabilità di NLC ha suggerito lo sviluppo di ETHO, sistemi di rilascio lipidici innovativi. Gli esperimenti in vitro su fibroblasti umani hanno rivelato il passaggio di ETHO attraverso la membrana cellulare e l'effetto dell'Ubichinone nella protezione da stress ossidativo. Infine, gel di Poloxamer sono stati formulati e caratterizzati per la veicolazione di Acido Gallico e Caffeico. I gel sono stati analizzati attraverso studi reologici e di spalmabilità, e sono stati eseguiti test in vitro per studiare la diffusione del principio attivo dai gel e la loro attività sulle cellule. Lo studio della struttura dei composti selezionati, nonché la loro interazione con le molecole attive e uno screening tecnologico, hanno permesso di sviluppare innovativi sistemi di drug delivery impiegabili in campo farmaceutico e cosmetico.The skin is the largest organ in the human body and it represents an important site for absorption of numerous molecules. Hence, the topical administration of drugs is an attractive way for the treatment of many local and systemic pathologies, displaying many advantages compared to the traditional oral and parenteral administration routes. Conventional topical preparations possess several limitations in the delivery of a drug molecule into systemic circulation, thus drug delivery systems characterized by micro and nano dimensions have been considered. The main advantages achieved with the development of novel carries are the targeted delivery and the enhancement of protection and stability of the encapsulated drug. Particularly, lipid-based delivery systems, thanks to their biodegradability, low toxicity and similarity with lipids composing epidermis represent promising tools for topical administration of drugs. In this doctorate project, Lipospheres (LS), Solid Lipid Nanoparticles (SLN), Nanostructured Lipid Carriers (NLC), Ethosomes (ETHO), and Poloxamer Gels have been proposed as new carrier systems for the topical administration of different active substances. Lipospheres Gel containing Clotrimazole have been described for the treatment of Candida albicans. The production method and the characterization in terms of size, morphology, encapsulation efficiency, viscosity and adhesion have been realized. Finally, in vitro anticandidal activity studies and release studies have been performed. Furthermore, a scaling up study focused on the production of Progesterone containing nanoparticles in a pilot scale have been investigated. Two different methods were developed and compared in order to obtain formulations in industrial scale. The study of dimension, morphology, encapsulation efficiency and stability of the produced nanoparticles has been realized. Moreover, the in vitro release and skin permeation studies have been performed by dialysis and Franz cells methods respectively, and the in vivo experiments have been conducted by tape stripping. The production of nanoparticle based gel have been designed to deliver Hyaluronic Acid and Retinyl Palmitate onto the skin for wound healing treatment, with the aim of generating a nanoparticulate gel suitable for cutaneous administration. In vitro wound healing have been performed on human cells in order to study the improvement of the drug activity when delivered by nanoparticulate gel. The production of lipid nanoparticles for -tocopherol delivery have been described with the aim of protecting human skin against pollutants, as cigarette smoke. Firstly, a preformulation study has been conducted to select the optimal nanoparticles composition for drug encapsulation. Then, the antioxidant effect of -tocopherol has been investigated though ex vivo experiments on human skin explants. NLC and ETHO containing Ubiquinone have been produced and compared, with the aim of developing a novel strategy in the treatment of Rett syndrome. The lack of stability of NLC-containing Ubiquinone suggested the development of drug-loaded ETHO, resulting as promising lipid-based delivery systems. In vitro experiments revealed the uptake of ETHO into human fibroblasts and the effect of Ubiquinone in protecting cells against oxidative insults. Finally, Poloxamer Gels have been formulated and characterized for the delivery of the antioxidants Gallic Acid and Caffeic Acid. The gels have been analyzed through rheological and spreadability assays, then in vitro tests have been performed to study the diffusion of the drug and the activity on cells. It has to be underlined the importance of a technological screening in the design of novel delivery nanosystems. The study of the components structure as well as their interaction with the interested actives allowed to develop different carrier systems by the employment of innovative techniques, suitable for both pharmaceutical and cosmetic fields

Antioxidant-containing monoolein aqueous dispersions: a preliminary study

: The present study describes a preliminary study on the use of monoolein aqueous dispersions (MADs) as delivery systems for antioxidant molecules, namely, ascorbyl palmitate (AP) and alpha-tocopherol (AT). MAD, produced by emulsifying monoolein (4.5% w/w) in water and poloxamer 407 (0.5% w/w) as emulsifier, was characterized in terms of size, morphology, and antioxidant activity by mean of PCS, cryo-TEM, and (2,2-diphenyl-1-picrylhydrazyl) assay. MAD-AP or MAD-AT gave rise to a bimodal size distribution with mean size around 200 nm. All the preparations stored at 25 °C showed quite stable size at least up to 90 days. Cryo-TEM images confirmed MAD size distribution and indicated different MAD morphologies as a function of the loaded antioxidant molecule. Indeed, in the case of MAD-AP, vesicles and cubosomes with the typical inner cubic structure were observed, while vesicles and hexosomes were shown for MAD-AT. The encapsulation efficiency of both antioxidants reached more than 90% with respect to the total amount of drug used for MAD preparation. Moreover, AP and AT antioxidant activity was retained after encapsulation, and in vitro Franz cell experiments showed that the MAD enabled to better control the drug release. These preliminary results suggest that MAD formulations could be further investigated as a potential delivery system for antioxidant supplementation in dietary or cosmetic fields

Nafion®-containing solid lipid nanoparticles as a tool for anticancer pt delivery: preliminary studies

Preliminary studies of nanoparticles based on the perfluorosulfonic acid resin Nafion® have been carried out with the aim of establishing an ionic connection with the protonable phosphine 1,3,5-triaza-7-phosphaadamantane (PTA), suitable for the coordination of platinum. Nafion®-containing nanoparticles (NAF) were produced by homogenization followed by ultrasonication method. After production, NAF were characterized in term of size, morphology and in vitro cytotoxicity. The PCS studies showed that the Z-Average mean diameter was around 250 nm. Moreover, the polydispersity index showed a monodimensional distribution of nanoparticles. Cryo-TEM analysis showed a uniform and homogenous population of particles, characterized by the presence of both ovoidal and needle-like structures. To evaluate the in vitro cytotoxicity, NAF were tested on human cancer cell lines K562 and A2780. No cytotoxic effect was found on both cell lines. By 13P-NMR measures, it is here proved that the strongly acidic sulfonic groups of Nafion®-containing nanoparticles (NAF) can act as protonating agents for PTA. The protonation occurs selectively at nitrogen, hence protonated PTA maintains its ability to coordinate platinum via its phosphorus atom