Lagreca, Elena (2023) Development of an innovative and versatile nano-emulsion- based platform for the active targeting. [Tesi di dottorato]

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Abstract

Nanoscale drug delivery systems represent a challenging area in pharmaceutical research, for the supply of therapeutics to the site of action, without affecting healthy tissues or organs. Herein, we report different strategies to engineer oil in water nano-emulsions (O/W NEs) to obtain a biomimetic nano-carrier employing both biological membrane and polymeric coating to increase the selective uptake into a selected compartment. Nowadays, a new bioengineered platform based on cell membrane cloaked nanoparticles has been developed to produce an innovative class of biomaterials able to combine the properties of cellular membranes with the engineering litheness of synthetic nanomaterials producing smart, biocompatible and biomimetic nanocarriers. This new class of cell membrane-camouflaged nanomaterials display numerous advantages such as high biocompatibility and prolonged blood circulation. Recently, we developed ultra-stable O/W NEs, able to carry both internal and external cargos (Somes) such as lipophilic compounds and hydrophilic coatings, respectively, that we call here NEsoSomes. O/W NEs are an excellent bioengineering tool for drug and molecules delivery due to their ability to dissolve a large number of hydrophobic compounds and protect them from hydrolysis and degradation under biological conditions. At present, no report is available on the combination of cell membrane coatings with such nanocarriers, probably due to their typical instability feature. Since that, we report for the first time a new cell membrane coated nanomaterial composed by membranes extracted from different cell lines including glioblastoma cancer cells (U87-MG) and THP-1 monocyte cell line deposited on NEsoSomes through a liquid–liquid interface method to produce highly controllable membrane coated nano-capsules. At first, we developed a biomimetic cancer cell based on secondary NEs (SNEs), named CM-NEsoSome, using as cell membrane source U87 glioblastoma cells as a suitable theranostic system to target cancer cells. CM-NEsoSomes were physiochemically characterized and in vitro validated. In detail, CM-NEsoSome biocompatibility was tested on a healthy model cell line, performing a cell cytotoxicity and uptake assay. The promising results of CM-NEsoSome led to the idea of building cell membrane coated nanoemulsions able to target inflamed tissue and more specifically atherosclerotic site. To target atherosclerotic lesions, we selected THP-1 as human monocyte cell line to obtain both human monocyte and macrophage membranes. THP-1 cells could be differentiated into macrophage-like phenotype cells (THP-1 macrophages, M0) by incubation with phorbol 12-myristate-13-acetate (PMA). Therefore, THP-1 were differentiated into macrophages to express cell membrane markers and exploit macrophage active targeting into atherosclerotic lesions. We developed two cell membrane coated NEsoSomes using THP-1(M) and PMA-differentiated THP-1(M0) as membrane sources of monocyte and an unpolarized macrophage, respectively. The two systems, indicated as Monocyte NEsoSome (M-NEsoSome) and Macrophage NEsoSome (M0-NEsoSome), joined together the biomimetic features coming from biological membrane with the physio-chemical and nano-sized characteristic of SNE. The uptake ability of two systems were evaluated both in a healthy endothelial cells (ECs) layer (represent by Human Umbilical Vein Endothelial Cells, HUVECs) and in a model of inflamed ECs obtained by treating HUVEC cell layer with Tumour Necrosis Factor α (TNFα). Interestingly, M0-NEsoSome demonstrated a pronounced accumulation in the inflamed ECs model as compared to the healthy cell layer demonstrating the selective targeting of this innovative type of nano-vector. Finally, a food grade oral drug delivery system has been proposed to provide a formulation with enhanced mucus-adhesion to the intestinal barrier. In this case, the nanocarrier is based on a secondary O/W NE prepared by adding thiol groups to chitosan (Ct) via a simple non-covalent procedure based on N-acetylcysteine (NAC) salification, which is compliant with food supplement formulations. Pharma grade and food grade formulations, in different materials combinations, were prepared and physio-chemically characterized (DLS, 1H-NMR, ITC, Cryo-TEM) showing similar behaviour. These systems were validated both in terms of bioaccessibility and mucus-adhesive properties employing respectively INFOGEST protocol and intestine on chip device (InOA-chip), which are able to mimic the complex intestinal functions. INFOGEST confirmed the improved bioaccessibility of encapsulated curcumin into our nano-system compared to the free molecule. Very interestingly, a significant enhancement in the mucus-adhesive properties of the proposed novel Ct-NAC NEs (due to the presence of thiol groups) was observed by comparing the two formulations behaviour through the InOA-chip. In conclusion, O/W SNEs demonstrate to be a suitable starting building block for a different type of oil based nanocarrier with biomimetic properties and selective accumulation in a precise compartment depending on the most external layer characteristics.

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