Research Institute for Medicines

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Publications

Molecular Insight into Mycobacterium tuberculosis Resistance to Nitrofuranyl Amides Gained through Metagenomics-like Analysis of Spontaneous Mutants

Publication . Mokrousov, Igor; Slavchev, Ivaylo; Solovieva, Natalia; Dogonadze, Marine; Vyazovaya, Anna; Valcheva, Violeta; Masharsky, Aleksey; Belopolskaya, Olesya; Dimitrov, Simeon; Zhuravlev, Viacheslav; Portugal, Isabel; Perdigão, João; Dobrikov, Georgi M.

We performed synthesis of new nitrofuranyl amides and investigated their anti-TB activity and primary genetic response of mycobacteria through whole-genome sequencing (WGS) of spontaneous resistant mutants. The in vitro activity was assessed on reference strain Mycobacterium tuberculosis H37Rv. The most active compound 11 was used for in vitro selection of spontaneous resistant mutants. The same mutations in six genes were detected in bacterial cultures grown under increased concentrations of 11 (2×, 4×, 8× MIC). The mutant positions were presented as mixed wild type and mutant alleles while increasing the concentration of the compound led to the semi-proportional and significant increase in mutant alleles. The identified genes belong to different categories and pathways. Some of them were previously reported as mediating drug resistance or drug tolerance, and counteracting oxidative and nitrosative stress, in particular: Rv0224c, fbiC, iniA, and Rv1592c. Gene-set interaction analysis revealed a certain weak interaction for gene pairs Rv1592–Rv1639c and Rv1592–Rv0224c. To conclude, this study experimentally demonstrated a multifaceted primary genetic response of M. tuberculosis to the action of nitrofurans. All three 11-treated subcultures independently presented the same six SNPs, which suggests their non-random occurrence and likely causative relationship between compound action and possible resistance mechanism.

2022-09-12Journal article

Open access

Solid Lipid Nanoparticles and Nanostructured Lipid Carriers as Smart Drug Delivery Systems in the Treatment of Glioblastoma Multiforme

Publication . Jnaidi, Raneem; Almeida, António José; Gonçalves, Lídia

Glioblastoma multiforme (GBM) is the most common and malignant type of brain tumor. In fact, tumor recurrence usually appears a few months after surgical resection and chemotherapy, mainly due to many factors that make GBM treatment a real challenge, such as tumor location, heterogeneity, presence of the blood-brain barrier (BBB), and others. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) represent the most promising carriers for therapeutics delivery into the central nervous system (CNS) owing to their inherent ability to cross the BBB. In this review, we present the main challenges in GBM treatment, a description of SLNs and NLCs and their valuable role as drug carriers in GBM treatment, and finally, a detailed description of all modification strategies that aim to change composition of SLNs and NLCs to enhance treatment outcomes. This includes modification of SLNs and NLCs to improve crossing the BBB, reduced GBM cell resistance, target GBM cells selectively minimizing side effects, and modification strategies to enhance SLNs and NLCs nose-to-brain delivery. Finally, future perspectives on their use are also be discussed, to provide insight about all strategies with SLNs and NLCs formulation that could result in drug delivery systems for GBM treatment with highly effective theraputic and minimum undesirable effects.

2020-09-10Journal article

Open access

Pyromellitic dianhydride crosslinked soluble cyclodextrin polymers: Synthesis, lopinavir release from sub-micron sized particles and anti-HIV-1 activity

Publication . Adeoye, Oluwatomide; Bártolo, Inês; Conceição, Jaime; Bento-Silva, Andreia; Duarte, Noélia; Francisco, Ana Paula; Taveira, Nuno; Marques, Helena Cabral

We report the synthesis of water soluble cyclodextrin (CD) polymers prepared by crosslinking pyromellitic dianhydride (PMDA) with two CD derivatives (methyl-β-CD - MβCD and (2-hydroxy)propyl-β-CD - HPβCD) and their evaluation as functional sub-micron sized carriers in the development of antiretroviral drug delivery systems. Using the protease inhibitor lopinavir (LPV) as model drug, LPV loaded CD polymers (pHPβCD and pMβCD) were prepared and fully characterized. The physicochemical characterization and in vitro drug release confirmed the successful synthesis of pHPβCD and pMβCD, the formation of sub-micron sized particles and a 12–14 fold increase in LPV solubility. Cytotoxicity assays indicated that both pHPβCD and pMβCD were able to improve the safety profile of LPV while the viral infectivity assay revealed a concentration independent anti-HIV-1 effect for both pHPβCD and pMβCD with a maximum percentage inhibition (MPI) of 79 and 91% respectively. After LPV loading, the antiviral profile of pHPβCD was reversed to the sigmoidal dose–response profile of LPV, while pMβCD maintained its dose-independent profile followed by a LPV mediated increase in viral inhibition. Overall, both pHPβCD and pMβCD demonstrated anti-HIV-1 activity, while drug loaded pMβCD indicated its potential as functional sub-micron sized drug delivery polymers for achieving synergistic anti-HIV activity.

2020-04Journal article

Restricted access

Targeting mitochondria of neural stem cells for neuroregeneration

Publication . Ribeiro, Maria Filipe Canas de Matos e Oliveira; Cruz, Susana Zeferino Solá da; Rodrigues, Cecília Maria Pereira

The adult mammalian brains display the capacity to generate new neurons from existing neural stem cells (NSCs) in a process called adult neurogenesis. This process drops sharply throughout ageing and is further impaired in neurodegenerative diseases and other neurological disorders. Curiously, mitochondria and mitochondrial metabolism have been shown to be key regulators of NSC fate. The influence of factors such as ageing, metabolism and diet on brain function is also becoming increasingly recognised. Interestingly, emerging data have supported a crosstalk between the brain and gut microbiota, through the modulation of host metabolism. In the present project, we aimed to dissect the molecular mitochondrial mechanisms responsible for neurogenesis alterations in the context of both neurodegeneration, such as in Alzheimer’s disease (AD), and dietary challenge. Initially, we investigated the impact of amyloid-β (Aβ) peptide, a hallmark of AD, in NSC fate and explored the contribution of mitochondria for Aβ-induced NSC changes. We showed that high levels of Aβ peptide result in mitochondrial signalling disruption, affecting NSC viability, proliferation and differentiation. Importantly, under elevated amyloid burden, an irreversible dysfunction of mitochondrial biogenesis, dynamics and oxidative state was found, precluding any rescue of neurogenesis through mitochondria. We then explored the role of mitochondrial signalling pathways in mediating the regulation of adult neurogenesis following a dietary challenge and subsequent changes in diet-associated gut microbiota. Interestingly, we discovered that animals fed a high-fat choline deficient diet (HFCD) diet display premature increased neurogenesis, which further exhausts the NSC pool for long-term neurogenesis. In fact, HFCD diet stimulated gut dysbiosis, upregulating metabolic pathways of short chain fatty acids (SCFAs), such as propionate and butyrate, in the small intestine and cecum. More importantly, the microbial metabolites enhanced mitochondrial biogenesis and oxidative stress in NSCs, while also promoting early neuronal differentiation through a ROS- and p-ERK1/2-dependent mechanism. Notably, this mitochondrial stressdependent pathway was activated in neurogenic niches of HFCD diet-fed mice. In conclusion, our findings clarify the impact of mitochondrial activity during adult neurogenesis and may prove useful in the development of novel strategies to rescue adult neurogenesis.

2019-10Doctoral thesis

Open access

Topical Administration of a Nanoformulation of Chitosan-Hyaluronic Acid-Epoetin Beta in a Rat Model of Glaucoma

Publication . Silva, Beatriz; Gonçalves, Lídia; Braz, Berta São; Delgado, Esmeralda

The present work investigates the effects of chitosan-hyaluronic acid-epoetin beta (CS/HA-EPOβ) nanoparticles after topical ocular administration in a rat glaucoma model. Wistar Hannover rats (n = 24) were submitted to a complete ophthalmological examination and electroretinography, followed by glaucoma induction in their right eye on day 1 of the study. Treatment group (T) received CS/HA-EPOβ nanocarriers (n = 12), while the control group (C) received only empty ones. Electroretinography was repeated on day 3 (n = 24) and before euthanasia on day 7 (n = 8), 14 (n = 8), and 21 (n = 8), followed by bilateral enucleation and histological assessment. The animals showed good tolerance to the nanoformulation. Maximum IOP values on the right eye occurred shortly after glaucoma induction (T = 62.6 ± 8.3 mmHg; C = 63.6 ± 7.9 mmHg). Animals from the treated group presented a tendency for faster recovery of retinal electrical activity (p > 0.05). EPOβ was detected on the retina of all treated eyes using immunofluorescence. Control animals presented with thinner retinas compared to the treated ones (p < 0.05). Therefore, topical ocular administration of CS/HA-EPOβ nanoparticles enabled EPOβ delivery to the retina of glaucomatous rats and promoted an earlier retinal recovery, confirming EPOβ’s neuroprotective effects. The encouraging results of this preclinical study pave the way for new strategies for topical ocular administration of neuroprotective compounds.

2023-01-23Journal article

Open access