Fabrication of in vitro epithelial tissues as a testing platform for drug delivery systems

In the last years, tissue engineering (TE) has advanced offering the potential for regenerating almost every tissue and organ of the human body. Three dimensional (3D) human tissue models can provide a good platform for pharmacological screening and drug delivery rather than traditional bidimensional (2D) cell culture or animal models. In native tissues the extracellular matrix (ECM) promotes the crosstalk between cells, and is responsible of several fundamental patophysiological processes. For this reason, tissue engineering is moving forward the development of in vitro models in which endogenous ECM is present. In the perspective of using in vitro tissues as testing platform. Nowadays there is an overlapping between tissue engineering and nanomedicine since they share the common application to improve the new molecules screening in vitro and the pre-clinical testing. Nanomedicine and nanofabrication allow to create miniaturized release-based systems such as nano-biocapsulates while novel tissue engineering processess allow to building up in vitro tissues and organ mimicking their in vivo counterpart. Nanomedicine relies on the fundamental principles of tissue engineering as well as the tissue engineering field favors from continuous progress in nanomedicine. Indeed, due to the complexity of these tissues, studying and analyzing possible strategies to implement specific nanocarriers is becoming more and more challenging. The big challenge is engenneering capsules able to specifically target a certain organ of interest and, then, ensure an effective in situ controlled release. 9 The aim of my PhD work is to effectively bridge nanomedicine and tissue engineering. Particularly, on the one hand-side, this work focused on the realization of functionalized nanoparticles that are able to transport lipophilic drug preventing their early degradation. On the other side, a 3D intestinal diseased model as a platform for drug screening was realized. We created nanocapsules starting from an oil-in-water (O/W) nanoemulsion coated with a polysaccharide layer film, i.e. a glycolmodified chitosan, and subsequently curcumin and paclitaxel were loaded. These compounds are usually unlikely water-soluble. In particular, curcumin can be involved as a factor for preventive therapy while Paclitaxel is included in farmaceuticals for treatment of colorectal carcinoma. The final nano-carriers are completely biocompatible and biostable. In the first part, I investigated the enhancement of the effect of curcumin loaded in our system across monolayers of intestinal epithelial cells (CaCo-2) in transwell culture. Such in vitro platform is suitable for evaluating the functionality of the nano-carrier and its specificity towards the mucosal epithelial layer. As an applicative example, the investigation of the anti-inflammatory effects exerted by the encapsulation of curcumin was carried out. In the second part, I developed a more complex in vitro cellular model. This is relevant for creating a unique 3D tumoral intestinal model capable of mimic physiological in vivo diseased architecture. Under these conditions, it was possible to precisely evaluate the antitumoral effets of paclitaxel in nanocapsules. In conclusion, I started from a 2D system to test the internalization capability and the efficacy of nanocapsules and then I moved towards 10 the development of a pathological 3D system and implemented the aforementioned internalization of nanocapsules loaded with potential chemioterapeutics (paclitaxel). Effectively, this work contributes to develop a future high throughput platform for drug screening of a variety of nano-carriers against tumoral-like cellular components.