Design and optimisation of drug delivery systems based on natural products for dermal and mucosal applications

Aim of the thesis was the design and optimization of nanodrug delivery systems based on natural products for dermal and mucosal application. Particularly, the described researches deal with the formulation of different lipid-nanocarriers: oil-in-water microemulsions, conventional liposomes, modified vesicles, like glycerosomes and propylene glycol-nanovesicles, and innovative vesicular systems called escinosomes and ascosomes. All these systems are very versatile and were in some cases loaded in hydrogels to increase their viscosity and/or mucoadhesion, as well as to provide semi-solid dosage forms which combine innovative and conventional technological approaches. Vesicles and microemulsions were prepared and optimized by different techniques and were physically and chemically characterized by dynamic/electrophoretic light scattering analyses, scanning/transmission electron microscopy and high performance liquid chromatography, in order to determine various technological parameters such as average hydrodynamic diameter, polydispersity index, ζ-potential, deformability, shape and encapsulation efficiency of loaded drugs, as well as to monitor the chemical and physical stability. In vitro release kinetics of the formulated drugs were evaluated by dialysis bag method and vertical diffusion Franz cells, using cellulose nitrate artificial membranes and suitable mathematical models. In vitro permeation performances were investigated by parallel artificial membrane permeation assay (PAMPA) and vertical diffusion Franz cells, using fresh excised rabbit ear skin , BALB/c nude mice skin and porcine buccal mucosa. Different cell lines were selected for in vitro activity or cytotoxicity studies. African Green Monkey kidney cells (Vero) infected with the herpes simplex virus type 1 were employed to evaluate the in vitro anti-herpetic activity of formulated substances, whereas different bacteria and fungi strains were used to test the in vitro antimicrobial activity. Cytotoxicity tests were done on immortalized human skin keratinocytes cell line (HaCaT). Uptake studies of drugs formulated in parenteral and oral dosage forms were carried out using parental and doxorubicin-resistant human erythroleukemia cells (K562, K562/DOXO), and Caco-2 cell line, respectively. In addition, in vivo experiments of acute dermal irritation/corrosion and acute dermal toxicity were carried out on Sprague Dawley rats, according to the OECD guidelines. In detail, a commercial saw palmetto carbon dioxide (CO2) extract was formulated in conventional liposomes, for the topical delivery of the active constituents and the potential usage in hair loss treatment. Glycerosomes, nanovesicles based on glycerol, were developed for loading three different Lamiaceae essential oils, i.e. Melissa officinalis L., Origanum onites L. and Satureja thymbra L., in order to protect the sensitive and volatile compounds from the direct exposure to environmental agents, as well as to provide a dosage form for dermal delivery of the essential oil avoiding skin irritancy. Since, vesicle bilayer can be made of a variety of amphiphilic molecules, innovative nanovesicular systems were explored in this thesis using bioactive molecules as vesicle bilayer forming components. In particular, escinosomes are escin-based vesicles, whereas ascosomes are ascorbyl derivatives-based vesicles. Both nanocarriers were developed for the skin delivery of the active bilayer constituents, as well as for loading and carrying further selected model drugs (berberine chloride and khellin, respectively). This thesis also aimed to develop two different microemulsion-based hydrogel (microemulgel) loaded with clobetasol propionate for the treatment of mucus membrane pemphigoid, a chronic vesicobullous dermatosis with autoimmune pathogenesis, and cannabidiol for the treatment of atopic dermatitis, a chronic relapsing condition characterized by itching and redness of the skin, widely spread among infants and children. Moreover, a novel microemulsion was designed for the dermatological application of khellin, selected as model natural drug because of its lipophilicity and anti-inflammatory properties. Both liposomes and microemulsions are very versatile systems, suitable for all the administration routes and also investigated in food-industry. Accordingly, a further study were focused on developing nanoliposomes loaded with berberine chloride and tariquidar, to enhance the intracellular uptake of the antitumoral drug in doxorubicin resistant human leukemia cells (K562/DOXO), thanks to the activity of the Pg-p inhibitor. Another investigation was focused on formulating essential oils of Origanum onites and Satureja thymbra in propylene glycol-nanovesicles, proposed as safe and effective delivery systems of alternative food preservatives. Lastly, a microemulsion for the oral delivery of an extract enriched of polyphenols from unripe olives (Olea europaea L.) was developed and in vitro fully characterized.