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  • New delivery systems based on gellan gum nanoparticles for Parkinson Diseases Therapeutics
    Publication . Rodrigues, Madalena Filipa Geada; Passarinha, Luís António Paulino; Sousa, Ângela Maria Almeida de; Cristóvão, Ana Clara Braz
    Globally, there are up to 10 million people diagnosed with Parkinson's Disease, a chronic neurodegenerative disorder without a cure. Physiologically, it is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Clinically, patients with this disease reveal several motor symptoms, such as tremors, tension, and postural instability, and non-motors, such as depression and anxiety. To date, the most effective drug combination used in the treatment of Parkinson's disease is the administration of levodopa (L-Dopa) combined with catechol-O-methyltransferase (COMT) inhibitors and monoamine oxidase inhibitors to restore dopaminergic brain levels. However, the commercially available inhibitors have low capabilities to cross the blood-brain barrier and, thus, low bioavailability in the brain. Also, the prolonged use of these drugs is associated with high hepatotoxicity, which currently limits their use. Therefore, the discovery of molecules with the potential to inhibit the COMT and the development of new delivery systems for these drugs are crucial elements to improve the effectiveness of existing therapies. Using polymeric nanoparticles as antiparkinsonian drug carriers presents numerous advantages considering the current therapies. These nanosystems can cross biological barriers due to their chemical properties and small size. Furthermore, they can achieve the therapeutic target more efficiently, increasing the bioavailability of the drug in restricted environments, such as the brain. Also, the application of polysaccharides in developing this type of delivery system presents advantages such as lower economic costs and better biocompatibility and biodegradability compared to oral and intravenous therapies. The discovery of the molecules 3,4-dihydroxy-5-nitrobenzonitrile (ZINC035) and 2- bromo-3,4-dihydroxy-S-nitrobenzaldehyde (ZINC496) as new COMT inhibitors, combined with the potential of nanoparticles based on natural polymers, such as gellan and chitosan, as drug delivery systems, have proven to be the impulse for this work. In the first part of this work, an analytical method using High-Performance Liquid Chromatography (HPLC) was developed to detect and quantify these new inhibitors in brain tissue samples from adult Wistar rats. In this study, the biological samples were spiked with each inhibitor and, subsequently, analyzed using two different types of detection, in which electrochemical detection (HPLC-ECD) was more effective for biological matrices than diode-array detection (HPLC-DAD). Additionally, a study was realized using different mobile phases, varying from 9 to 18% (v/v) of the organic compound (acetonitrile) to optimize the experimental procedure time. The results obtained using an HPLC-ECD analysis system interestingly showed that the molecules under study have interaction capabilities with brain tissues, and due to their similarity with commercial inhibitors, their potential use in Parkinson's therapies has been proven. In the progress of this work, a new polymeric delivery system has been developed to improve metabolism and absorption, increasing their chemical stability, decreasing their susceptibility to enzymatic degradation, and improving bioavailability of the commercial drugs. This delivery system results from the complexation of two natural polymers with opposite charges, the gellan gum (GG) and the chitosan (CH). These polymeric complexes were formulated, testing different conditions, varying the molecular weight of chitosan (5 kDa and low molecular weight) and the ratio and concentration of the polymers (0.05-1 mg/mL). After optimization, the GG/CH systems were prepared with the inclusion of the selected COMT inhibitor (ZINC035) or L-Dopa. In order to obtain the encapsulation efficiency of the nanoparticles, a purification system was developed, testing three different methodologies (centrifugation, filtration and molecular exclusion chromatography) and varying some conditions, such as centrifugation speeds (8000- 12500 rpm), the size of filter pore (0.22 and 0.45 µm) and the elution buffers (sodium acetate, sodium chloride and phosphate-buffered saline). The best GG/CH delivery system had a size of 238.52 nm; a polydispersity index (PDI) value of 0.449 and a zeta potential (ZP) of +30.2 mV. Also, GG/CH nanoparticles loaded with ZINC035 showed a mean size of 163.5 nm; a PDI of 0.355; a zeta potential of +20.6 mV and an encapsulation efficiency of 67.04%, which are stable up to 48h after formulation. Finally, the L-Dopa-loaded delivery systems achieved a size of 177.01 nm, a PDI of 0.392, and an encapsulation efficiency of approximately 26%. Overall, the HPLC system with electrochemical detection developed in this work is an effective and innovative methodology for studying COMT inhibitors in biological tissues. Furthermore, nanoparticulate polymeric systems have unique properties that can be used according to the desired intranasal delivery application and will certainly present advantages over conventional therapies.
    2025-02-12Master thesis Embargoed access Show more