• biomaterials
• melt-spinning
• naringenin
• PHBV
• PLGA
• wet-spinning
• drug delivery
• inflammation
• LPS
• BV2
• RT qPCR
• immunofluorescence

Neurodegenerative diseases of the retina are one of the leading causes of impaired vision and blindness worldwide. Oxidative stress is considered as the triggering factor of this type of diseases, as it determines the chronic activation of the pro-inflammatory phenotype of microglia. Microglia are the cell type responsible for the immune protection of the central nervous system, and, in retinopathies, their impaired functioning triggers neurological damage that progresses toward deterioration of the retina up to complete loss of vision.
Flavonoids, molecules of natural origin, have been proposed as active ingredients to prevent and protect against the effects of this pathology due to their antioxidant and anti-inflammatory activity. In particular, flavonoid naringenin has been tested in in vivo models of retinopathies, proving to be very effective in counteracting oxidative stress and inflammation. However, the most common method for the treatment of this kind of pathologies, intravitreal injection, is often inefficient for many formulations, as well as being an invasive system and presenting possible complications for the patient.
This thesis project proposes the production of controlled release systems of naringenin and the evaluation of their effects in vitro on the murine microglia cell line (BV2). Cylindrical-shaped polymeric matrix devices, referred to as rods, were fabricated, physical-chemically characterized, and in vitro tested as potential intravitreal implants in the treatment of patients affected by retinopathies.
The materials chosen for these devices are poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), synthesized by different species of bacteria, therefore obtained by sustainable processes, and poly(lactide-co-glycolide) (PLGA), chosen to facilitate the processing of PHBV and because it is already authorized in Europe and the United States for biomedical applications. Two different types of rod were produced, by means of different processing techniques (i.e., wet-spinning and melt-spinning) with a 5% w/w theoretical concentration of naringenin. The devices loaded with the active principle were analyzed by means of scanning electron microscopy which allowed us to define the differences in morphology determined by the selected processing techniques. The rods were also characterized from a thermal point of view by thermogravimetric analysis technique and differential scanning calorimetry. The flavonoid was also extracted from the devices with a selective solvent to establish the actual amount of naringenin encapsulated within the polymeric matrix. All these analyses were compared to rods previously produced by means of the same techniques but with a naringenin content of 1%.
The release kinetics of the flavonoid was then examined by rods incubation in phosphate buffer saline (PBS) at 37 °C and UV spectroscopy analysis, and the results were used as a basis for in vitro experiments on BV2 cells, a microglia line.
The compatibility of the devices on the cell line and the ability to release naringenin in the culture medium were thus tested. To this aim, BV2 cells were pre-treated with medium previously incubated with loaded devices in order to promote the release of a desired concentration of the flavonoid or in direct contact with the devices. After six hours, an inflammatory response was induced in vitro, by incubation with bacterial lipopolysaccharide (LPS). Finally, through Real Time qPCR, the ability of the different treatments to reduce the expression levels of genes considered indicators of inflammation was investigated: interleukin 1 beta (IL-1 β), interleukin 6 (IL-6) and the C-C Motif Chemokine Ligand 2 (CCL2).