Browsing by Author "Neto, Miguel Leite"
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- Formulation and characterization of taxifolin delivery systems as an anti-Human Papillomavirus drugPublication . Neto, Miguel Leite; Sousa, Ângela Maria Almeida de; Costa, Diana Rita BarataCancer is a leading cause of mortality worldwide, with over 19.3 million cases reported in 2020, resulting in approximately 10 million deaths. Various risk factors have been associated with the development of cancer, including age, genetic predisposition, ethnicity, environmental exposure, lifestyle, and infections caused by bacteria, parasites, or viruses. Human papillomavirus (HPV) is the primary causative agent of cervical cancer, which is the fourth most common cancer in women worldwide, responsible for over 340,000 deaths in 2020. This cancer is characterized by the overexpression of oncoproteins E6 and E7, which disrupt cell cycle regulation and proliferation, compromising the functions of tumor suppressor proteins p53 and pRb, respectively. Despite the availability of preventive HPV vaccines, their administration is not universally carried out. Consequently, efforts continue to develop effective therapies for this type of cancer to achieve a successful cure. In this regard, flavonoids have demonstrated remarkable therapeutic potential, offering an accessible and effective approach, allowing their use in less developed countries with limited healthcare resources. Taxifolin is a flavonoid with several anticancer properties, having shown particular relevance in cervical cancer therapy due to its ability to enhance the inhibition of oncoprotein E6, thereby increasing p53 expression and inducing apoptosis. However, the use of taxifolin is limited due to its very low aqueous solubility and low stability. Consequently, its poor bioavailability restricts its applications in cancer therapies. Thus, this master's dissertation aims to develop delivery systems for the encapsulation of taxifolin to improve its bioavailability and effectiveness in HPV-positive cells. To achieve this, various delivery systems consisting of chitosan, gelatin, and taxifolin were formulated. Initially, several ratios of systems composed solely of low molecular weight chitosan and gellan gum were tested. The most favourable ratio exhibited a size of 238.07 ± 31.18 nm, a PdI of 0.29 ± 0.08, and a zeta potential of +22.56 ± 2.83 mV and was chosen to proceed with the studies and attempt taxifolin encapsulation. For the incorporation of taxifolin into the delivery systems, various types of chitosan (5 kDa, low molecular weight, and high molecular weight) were tested at various taxifolin ratios. Systems composed of low molecular weight chitosan (LMW CH/GG/TAX) had a size of 276.23 ± 30.68 nm, a PdI of 0.36 ± 0.06, and a zeta potential of +30.80 ± 5.66 mV. Delivery systems formed by high molecular weight chitosan (HMW CH/GG/TAX) showed a size of 272.82 ± 53.58 nm, a PdI of 0.33 ± 0.10, and a zeta potential of +23.59 ± 5.94 mV, while systems composed of 5 kDa chitosan (5 kDa CH/GG/TAX) had a size of 249.00 ± 12.58 nm, a PdI of 0.35 ± 0.10, and a zeta potential of +17.35 ± 5.64 mV. Regarding encapsulation efficiency, LMW CH/GG/TAX exhibited an efficiency of 65%, while the remaining systems encapsulated only 55%. Scanning electron microscopy (SEM) was also performed for all formulated samples, and it was possible to observe that all systems exhibited a spherical and uniform morphology. In addition, Fourier-transform infrared spectroscopy (FTIR) and UV/vis spectroscopy were conducted to verify the presence and interactions of compounds in the delivery systems. Finally, in vitro release assays were performed at two different pH levels, one representative of the tumor microenvironment (pH 5.8) and one representative of physiological pH (pH 7.4). The results showed that HMW CH/GG/TAX systems released approximately 45% and 80% of taxifolin at pH 5.8 and pH 7.4, respectively. LMW CH/GG/TAX systems released 25% at pH 7.4 and approximately 70% at pH 5.8. The 5 kDa CH/GG/TAX systems released between 20% and 40% at the two different pH levels. Based on all the gathered information regarding the characterization of the delivery systems, only the most favourable formulation, LMW CH/GG/TAX, proceeded to cellular assays, where internalization assays were conducted on healthy cells and HPV-positive cells. The results demonstrated that the delivery systems were capable of internalizing into both cell lines; however, it is expected that they will solely exert an effect on HPV-positive cells due to their specific action against oncoprotein E6.