Naturally-Derived, Bioactive, Zein Microparticles for Wound Healing: Efficiency Optimization via Spray Drying

Wound healing is a delicate process comprising various phases, of which inflammation constitutes the most critical one. Some wounds fail to evolve from this state due to pre-existing pathologies or medical conditions, such as diabetes, becoming chronic and causing pain and serious consequences for the patients. Wounds are defined chronic when they fail to heal in a time span that ranges from four weeks to three months and, as an extreme consequence, can eventually lead to gangrene and patient’s death, as it may happen with severe cases of diabetes. Chronic wounds represent an enormous economic burden for society: for instance, NHS reported that in 2019 there were circa 739 000 leg ulcers in England, with estimated healthcare costs of £3 billion per annum. Given these premises, it is imperative to (i) design innovative materials able to support the patient’s body in the transition through the inflammatory and the proliferative phases, as well as to (ii) define and optimize fabrication protocols able to strengthen the functional materials therapeutic efficiency. In the first part of this thesis, a method to produce zein microparticles (ZμPs) loaded with chemically modified curcumin (mCUR), a novel bioactive molecule with strong antioxidant properties, is described. Such functional microstructures are envisioned to be delivered to the wound site, to help in resolving the inflammatory state. Derived from the classic curcumin, mCUR chemical structure has been modified with an amino phenyl carbonyl group on C4 to (i) overcome the poor bioavailability in the human body, and to (ii) improve its inhibitory activity on the inflammatory markers thanks to a higher in vitro efficacy (lower IC50). ZμPs, both empty and loaded, were fabricated via the spray drying technique, using the two-fluid-nozzle modality (2FN), starting from various zein/mCUR ratios (2FN mCUR_ZμPs). An exhaustive characterization of the physico-chemical properties of the 2FN microparticles, either empty or loaded, is presented. Their biocompatibility was tested on human keratinocytes and their bioactivity in terms of in vitro antioxidant properties was assessed via the DCFH-DA assay, where a fluorescence decrease proportional to mCUR concentration was clearly visible (down to 41.7 %), highlighting the potential benefit of our design. Finally, preliminary antibacterial properties of mCUR_ZμPs were assessed on Escherichia coli cultures. With the aim of further enhancing the mCUR_ZμPs features in terms of drug loading, drug encapsulation efficiency and drug release profile, in the second part of this thesis work mCUR_ZμPs were fabricated employing the three-fluid-modality of the spray dryer (3FN mCUR_ZμPs). From this optimization step, the encapsulation efficiency was noticeably improved, reaching values close to 100 % (99.9 ± 0.04 % for the highest zein/mCUR ratio). This result was consistent with zeta potential analysis, which showed how the isoelectric point of the 3FN mCUR_ZμPs was closer to that of empty ZμPs, suggesting the achievement of a complete and more stable core/mCUR – shell/zein structure. Moreover, 3FN mCUR_ZμPs were characterized by a slower drug release, that allowed for a stronger antioxidant activity of mCUR when tested in vitro on human keratinocytes (fluorescence intensity dropped down to 31.1 %). Focus of the last part of the thesis was the design of a composite 3D hydrogel network, based on an injectable combination of collagen and gelatin, loaded with either 2FN mCUR_ZμPs or 3FN mCUR_ZμPs, to better modulate the mCUR release profile. Our formulations successfully reduced oxidative stress inside the cells thanks to a gradual release of the active molecule, especially when produced with the three-fluid-modality. The optimized fabrication process supports the realization of ad hoc microparticulate powders, to be applied for specific applications for wound healing purposes. Moreover, we investigated the possibility to incorporate them into advanced systems suitable to be applied directly on the wound bed to finely vehicle the release of mCUR in situ. Overall, our naturally derived microparticles showed promising results for future in vivo testing, in order to fully exploit their beneficial effects, and successfully become a valuable therapeutic option for chronic wound patients.