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Abdulmawjood, B, Roma-Rodrigues C, Fernandes AR, Baptista PV.  2019.  Liquid biopsies in myeloid malignancies, 2019. Cancer Drug Resistance. 2(4):1044-1061. Abstract

Hematologic malignancies are the most common type of cancer affecting children and young adults, and encompass diseases, such as leukemia, lymphoma, and myeloma, all of which impact blood associated tissues such as the bone marrow, lymphatic system, and blood cells. Clinical diagnostics of these malignancies relies heavily on the use of bone marrow samples, which is painful, debilitating, and not free from risks for leukemia patients. Liquid biopsies are based on minimally invasive assessment of markers in the blood (and other fluids) and have the potential to improve the efficacy of diagnostic/therapeutic strategies in leukemia patients, providing a useful tool for the real time molecular profiling of patients. The most promising noninvasive biomarkers are circulating tumor cells, circulating tumor DNA, microRNAs, and exosomes. Herein, we discuss the role of assessing these circulating biomarkers for the understanding of tumor progression and metastasis, tumor progression dynamics through treatment and for follow-up.

Almeida, J, Roma-Rodrigues C, Mahmoud AG, Guedes da Silva MFC, Pombeiro AJL, Martins LMDRS, Baptista PV, Fernandes AR.  2019.  Structural characterization and biological properties of silver(I) tris(pyrazolyl)methane sulfonate, 2019. J Inorg Biochem. 199:110789. AbstractWebsite

The water-soluble 1D helical coordination polymer [Ag(Tpms)]n (1) [Tpms=tris(pyrazolyl)methane sulfonate, (-)O3SC(pz)3; pz=pyrazolyl] was synthesized and fully characterized, its single-crystal X-ray diffraction analysis revealing the ligand acting as a bridging chelate N3-donor ligand. The antiproliferative potential of 1 was performed on two human tumour cell lines, A2780 and HCT116, and in normal fibroblasts, with a much higher effect in the former cell line (IC50 of 0.04muM) as compared to the latter cell line and to normal fibroblasts. Compound 1 does not alter cell cycle progression but interferes with the adherence of A2780 cells triggering cell apoptosis. Apoptosis appears to occur via the extrinsic pathway (no changes in mitochondria membrane potential, reactive oxygen species (ROS) and pro-apoptotic (B-cell lymphoma 2 (BCL-2) associated protein (BAX))/anti-apoptotic (BCL-2) ratio) being this hypothesis also supported by the presence of silver mainly in the supernatants of A2780 cells. Results also indicated that cell death via autophagy was triggered. Proteomic analysis allowed us to confirm that compound 1 is able to induce a stress response in A2780 cells that is related with its antiproliferative activity and the trigger of apoptosis.

Alves, PU, Vinhas R, Fernandes AR, Birol SZ, Trabzon L, Bernacka-Wojcik I, Igreja R, Lopes P, Baptista PV, Aguas H, Fortunato E, Martins R.  2018.  Multifunctional microfluidic chip for optical nanoprobe based RNA detection - application to Chronic Myeloid Leukemia, 2018. Sci Rep. 8(1):381. AbstractWebsite

Many diseases have their treatment options narrowed and end up being fatal if detected during later stages. As a consequence, point-of-care devices have an increasing importance for routine screening applications in the health sector due to their portability, fast analyses and decreased cost. For that purpose, a multifunctional chip was developed and tested using gold nanoprobes to perform RNA optical detection inside a microfluidic chip without the need of molecular amplification steps. As a proof-of-concept, this device was used for the rapid detection of chronic myeloid leukemia, a hemato-oncological disease that would benefit from early stage diagnostics and screening tests. The chip passively mixed target RNA from samples, gold nanoprobes and saline solution to infer a result from their final colorimetric properties. An optical fiber network was used to evaluate its transmitted spectra inside the chip. Trials provided accurate output results within 3 min, yielding signal-to-noise ratios up to 9 dB. When compared to actual state-of-art screening techniques of chronic myeloid leukemia, these results were, at microscale, at least 10 times faster than the reported detection methods for chronic myeloid leukemia. Concerning point-of-care applications, this work paves the way for other new and more complex versions of optical based genosensors.

Alves, PU, Vinhas R, Fernandes AR, Birol SZ, Trabzon L, Bernacka-Wojcik I, Igreja R, Lopes P, Baptista PV, Águas H, Fortunato E, Martins R.  2018.  Multifunctional microfluidic chip for optical nanoprobe based RNA detection - application to Chronic Myeloid Leukemia, 2018. Scientific reports. 8(1):381. Abstract
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Alves Ferreira, D, L MMDRS, A FR, Martins M.  2020.  A Tale of Two Ends: Repurposing Metallic Compounds from Anti-Tumour Agents to Effective Antibacterial Activity, 2020. Antibiotics (Basel). 9(6) AbstractWebsite

The rise in antibiotic resistance coupled with the gap in the discovery of active molecules has driven the need for more effective antimicrobials while focusing the attention into the repurpose of already existing drugs. Here, we evaluated the potential antibacterial activity of one cobalt and two zinc metallic compounds previously reported as having anticancer properties. Compounds were tested against a range of Gram-positive and -negative bacteria. The determination of the minimum inhibitory and bactericidal concentrations (MIC/MBC) of the drugs were used to assess their potential antibacterial activity and their effect on bacterial growth. Motility assays were conducted by exposing the bacteria to sub-MIC of each of the compounds. The effect of sub-MIC of the compounds on the membrane permeability was measured by ethidium bromide (EtBr) accumulation assay. Cell viability assays were performed in human cells. Compound TS262 was the most active against the range of bacteria tested. No effect was observed on the motility or accumulation of EtBr for any of the bacteria tested. Cell viability assays demonstrated that the compounds showed a decrease in cell viability at the MIC. These results are promising, and further studies on these compounds can lead to the development of new effective antimicrobials.

Alves-Barroco, C, Roma-Rodrigues C, Balasubramanian N, Guimaraes MA, Ferreira-Carvalho BT, Muthukumaran J, Nunes D, Fortunato E, Martins R, Santos-Silva T, Figueiredo AMS, Fernandes AR, Santos-Sanches I.  2019.  Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae, 2019. Int J Med Microbiol. 309(3-4):169-181. AbstractWebsite

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

Alves-Barroco, C, Roma-Rodrigues C, Raposo LR, Bras C, Diniz M, Caco J, Costa PM, Santos-Sanches I, Fernandes AR.  2019.  Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models, 2019. Microbiologyopen. 8(1):e00623. AbstractWebsite

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.

Alves-Barroco, C, Paquete-Ferreira J, Santos-Silva T, Fernandes AR.  2020.  Singularities of Pyogenic Streptococcal Biofilms – From Formation to Health Implication, 2020. 11(3179) AbstractWebsite

Biofilms are generally defined as communities of cells involved in a self-produced extracellular matrix adhered to a surface. In biofilms, the bacteria are less sensitive to host defense mechanisms and antimicrobial agents, due to multiple strategies, that involve modulation of gene expression, controlled metabolic rate, intercellular communication, composition, and 3D architecture of the extracellular matrix. These factors play a key role in streptococci pathogenesis, contributing to therapy failure and promoting persistent infections. The species of the pyogenic group together with Streptococcus pneumoniae are the major pathogens belonging the genus Streptococcus, and its biofilm growth has been investigated, but insights in the genetic origin of biofilm formation are limited. This review summarizes pyogenic streptococci biofilms with details on constitution, formation, and virulence factors associated with formation.

Alves-Barroco, C, Rivas-Garcia L, Fernandes AR, Baptista PV.  2020.  Tackling Multidrug Resistance in Streptococci - From Novel Biotherapeutic Strategies to Nanomedicines, 2020. Front Microbiol. 11:579916. AbstractWebsite

The pyogenic streptococci group includes pathogenic species for humans and other animals and has been associated with enduring morbidity and high mortality. The main reason for the treatment failure of streptococcal infections is the increased resistance to antibiotics. In recent years, infectious diseases caused by pyogenic streptococci resistant to multiple antibiotics have been raising with a significant impact to public health and veterinary industry. The rise of antibiotic-resistant streptococci has been associated to diverse mechanisms, such as efflux pumps and modifications of the antimicrobial target. Among streptococci, antibiotic resistance emerges from previously sensitive populations as result of horizontal gene transfer or chromosomal point mutations due to excessive use of antimicrobials. Streptococci strains are also recognized as biofilm producers. The increased resistance of biofilms to antibiotics among streptococci promote persistent infection, which comprise circa 80% of microbial infections in humans. Therefore, to overcome drug resistance, new strategies, including new antibacterial and antibiofilm agents, have been studied. Interestingly, the use of systems based on nanoparticles have been applied to tackle infection and reduce the emergence of drug resistance. Herein, we present a synopsis of mechanisms associated to drug resistance in (pyogenic) streptococci and discuss some innovative strategies as alternative to conventional antibiotics, such as bacteriocins, bacteriophage, and phage lysins, and metal nanoparticles. We shall provide focused discussion on the advantages and limitations of agents considering application, efficacy and safety in the context of impact to the host and evolution of bacterial resistance.

Amendoeira, A, García LR, Fernandes AR, Baptista PV.  2020.  Light Irradiation of Gold Nanoparticles Toward Advanced Cancer Therapeutics, 2020. 3(1):1900153. AbstractWebsite

Abstract Cancer is one of the leading causes of death in the world. To challenge this epidemic, there are growing demands for the development of new advanced and targeted therapeutics capable of effectively tackling cancer cells with improved selectivity. Nanomedicine has put forward several innovative therapeutics toward improving therapeutic efficacy while decreasing the deleterious side effects of current chemotherapy. Multifunctional gold nanoparticles (AuNPs) have been at the core of a plethora of advanced therapeutic strategies that provide selective targeting with their unique optical properties, capable to interact with the light of specific wavelength to deliver therapy with tremendous spatiotemporal precision. AuNPs have been exploited as photodynamic and photothermal therapeutic agents alone or in combination with other cancer treatment modalities with other cancer applications. Due to their exceptional physicochemical properties, they have been proven efficacious allies for photodynamic therapy and for photothermal therapy regimens. Herein, the rapidly progressing literature related to the use of these promising strategies against cancer is discussed, highlighting their possible future clinical translation.

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Baptista, PV, McCusker MP, Carvalho A, Ferreira DA, Mohan NM, Martins M, Fernandes AR.  2018.  Nano-Strategies to Fight Multidrug Resistant Bacteria—“A Battle of the Titans”, 2018. 9(1441) AbstractWebsite

Infectious diseases remain one of the leading causes of morbidity and mortality worldwide. The WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. Therefore, the antibiotic resistance crisis is one of the most pressing issues in global public health. Associated with the rise in antibiotic resistance is the lack of new antimicrobials. This has triggered initiatives worldwide to develop novel and more effective antimicrobial compounds as well as to develop novel delivery and targeting strategies. Bacteria have developed many ways by which they become resistant to antimicrobials. Among those are enzyme inactivation, decreased cell permeability, target protection, target overproduction, altered target site/enzyme, increased efflux due to over-expression of efflux pumps, among others. Other more complex phenotypes, such as biofilm formation and quorum sensing do not appear as a result of the exposure of bacteria to antibiotics although, it is known that biofilm formation can be induced by antibiotics. These phenotypes are related to tolerance to antibiotics in bacteria. Different strategies, such as the use of nanostructured materials, are being developed to overcome these and other types of resistance. Nanostructured materials can be used to convey antimicrobials, to assist in the delivery of novel drugs or ultimately, possess antimicrobial activity by themselves. Additionally, nanoparticles (e.g., metallic, organic, carbon nanotubes, etc.) may circumvent drug resistance mechanisms in bacteria and, associated with their antimicrobial potential, inhibit biofilm formation or other important processes. Other strategies, including the combined use of plant-based antimicrobials and nanoparticles to overcome toxicity issues, are also being investigated. Coupling nanoparticles and natural-based antimicrobials (or other repurposed compounds) to inhibit the activity of bacterial efflux pumps; formation of biofilms; interference of quorum sensing; and possibly plasmid curing, are just some of the strategies to combat multidrug resistant bacteria. However, the use of nanoparticles still presents a challenge to therapy and much more research is needed in order to overcome this. In this review, we will summarize the current research on nanoparticles and other nanomaterials and how these are or can be applied in the future to fight multidrug resistant bacteria.

Bathula, C, Roma-Rodrigues C, Chauhan J, Fernandes AR, Sen S.  2018.  Synthesis of tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolones via intramolecular oxidative ring rearrangement of tetrahydro-β-carbolines and their biological evaluation, 2018. New Journal of Chemistry. 42(8):6538-6547. AbstractWebsite

A simple oxidative ring rearrangement of diversely substituted 1-(2-amminoaryl)-tetrahydro-β-carbolines has been developed to generate architecturally interesting tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolones. This unique transformation involves four reaction centers (aniline, C1-carboline and C2/C3 of indole) and utilizes tert-butylhypochlorite as the reagent. The generic nature of the reaction was demonstrated by the synthesis of a wide variety of analogs 9a–j. A putative reaction mechanism was proposed. Cytotoxicity screening of these compounds against three human cancer cells (A2780 ovarian and HCT116 colorectal carcinoma cell lines and A549 lung adenocarcinoma cell line) revealed selective inhibition of proliferation of the A2780 human ovarian carcinoma cell line by one of the molecules 9a with an IC50 of 14 μM. No cytotoxic activity was observed in human normal fibroblasts for concentrations up to 100 μM. Compound 9a induced hyperpolarization of the mitochondrial membrane potential of the A2780 cell line leading to an increase of reactive oxygen species (ROS) that trigger cell death via apoptosis. Interestingly, compound 9a was also able to induce cell death via autophagy. Compounds that induce apoptosis and autophagy, thus leading to cancer cells’ death, are good candidates for cancer therapy.

Beola, L, Asin L, Roma-Rodrigues C, Fernandez-Afonso Y, Fratila RM, Serantes D, Ruta S, Chantrell RW, Fernandes AR, Baptista PV, de la Fuente JM, Grazu V, Gutierrez L.  2020.  The Intracellular Number of Magnetic Nanoparticles Modulates the Apoptotic Death Pathway after Magnetic Hyperthermia Treatment, 2020. ACS Appl Mater Interfaces. 12(39):43474-43487. AbstractWebsite

Magnetic hyperthermia is a cancer treatment based on the exposure of magnetic nanoparticles to an alternating magnetic field in order to generate local heat. In this work, 3D cell culture models were prepared to observe the effect that a different number of internalized particles had on the mechanisms of cell death triggered upon the magnetic hyperthermia treatment. Macrophages were selected by their high capacity to uptake nanoparticles. Intracellular nanoparticle concentrations up to 7.5 pg Fe/cell were measured both by elemental analysis and magnetic characterization techniques. Cell viability after the magnetic hyperthermia treatment was decreased to <25% for intracellular iron contents above 1 pg per cell. Theoretical calculations of the intracellular thermal effects that occurred during the alternating magnetic field application indicated a very low increase in the global cell temperature. Different apoptotic routes were triggered depending on the number of internalized particles. At low intracellular magnetic nanoparticle amounts (below 1 pg Fe/cell), the intrinsic route was the main mechanism to induce apoptosis, as observed by the high Bax/Bcl-2 mRNA ratio and low caspase-8 activity. In contrast, at higher concentrations of internalized magnetic nanoparticles (1-7.5 pg Fe/cell), the extrinsic route was observed through the increased activity of caspase-8. Nevertheless, both mechanisms may coexist at intermediate iron concentrations. Knowledge on the different mechanisms of cell death triggered after the magnetic hyperthermia treatment is fundamental to understand the biological events activated by this procedure and their role in its effectiveness.

Bravo, C, Robalo PM, Marques F, Fernandes AR, Sequeira DA, M. Piedade FM, Garcia HM, de Brito MVJ, Morais TS.  2019.  First heterobimetallic Cu(i)–dppf complexes designed for anticancer applications: synthesis, structural characterization and cytotoxicity, 2019. New Journal of Chemistry. 43(31):12308-12317. AbstractWebsite

A new family of eight heterobimetallic Cu(i)–dppf complexes of general formula [Cu(dppf)L][BF4] with dppf = 1,1′-bis(diphenylphosphino)ferrocene and L representing N,N-, N,O- and N,S-heteroaromatic bidentate ligands have been synthesized and fully characterized by classical analytical, spectroscopic and electrochemical methods. The single crystal structures of [Cu(dppf)(pBI)][BF4] (6), [Cu(dppf)(dpytz)][BF4] (7) and [Cu(dppf)(5-Aphen)][BF4] (8) complexes (where pBI = 2-(2-pyridyl)benzimidazole, dpytz = 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine and 5-Aphen = 1,10-phenanthrolin-5-amine) were determined by X-ray diffraction studies. Cytotoxicity of all complexes was evaluated in two human breast adenocarcinoma cell lines (MCF7 and MDAMB231). All the complexes exhibit high cytotoxicity against both human breast cancer cells with IC50 values far lower than those found for the antitumor drug cisplatin in the same cell lines. The IC50 values on primary healthy fibroblasts are of the same order of magnitude as those found for the tumoral cells.

Busila, M, Tabacaru A, Mussat V, Vasile BS, Neasu IA, Pinheiro T, Roma-Rodrigues C, Baptista PV, Fernandes AR, Matos AP, Marques F.  2020.  Size-Dependent Biological Activities of Fluorescent Organosilane-Modified Zinc Oxide Nanoparticles, 2020. J Biomed Nanotechnol. 16(2):137-152. AbstractWebsite

Surface modification of zinc oxide nanoparticles (ZnO NPs) is a strategy to tune their biocompatibility. Herein we report on the synthesis of a series of fluorescent ZnO NPs modified with 2-10% (3-glycidyloxypropyl)trimethoxysilane (GPTMS) to investigate the fluorescence properties and to explore their applications in microbiology and biomedicine. The obtained ZnO NPs were characterized by X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). Size reduction occurred from ca. 13 nm in unmodified ZnO to 3-4 nm in silane-modified samples and fluorescence spectra showed size-dependent variation of the photoemission bands' intensity. The antibacterial and cytotoxic activities were investigated on Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, and in ovarian (A2780) and prostate (PC3) cancer cells by tetrazolium/formazan-based methods. The antibacterial effect was higher for E. coli than S. aureus, while the cytotoxic activity was similar for both cancer cells and varied with the particle size. Cell death by apoptosis, and/or necrosis versus autophagy, were explored by flow cytometry using an Annexin V based-method and transmission electron microscopy (TEM). The main mechanism of ZnO NPs toxicity may involve the generation of reactive oxygen species (ROS) and the induction of apoptosis or autophagy. This work revealed the potential utility of GPTMS-modified ZnO NPs in the treatment of bacterial infection and cancer.

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Chohan, ZH, Shad HA, Supuran CT.  2012.  Synthesis, characterization and biological studies of sulfonamide Schiff’s bases and some of their metal derivatives, 2012. Journal of Enzyme Inhibition and Medicinal ChemistryJournal of Enzyme Inhibition and Medicinal Chemistry. 27(1):58-68.: Taylor & Francis AbstractWebsite
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Choroba, K, Raposo LR, Palion-Gazda J, Malicka E, Erfurt K, Machura B, Fernandes AR.  2020.  In vitro antiproliferative effect of vanadium complexes bearing 8-hydroxyquinoline-based ligands - the substituent effect, 2020. Dalton Trans. 49(20):6596-6606. AbstractWebsite

This is the first comprehensive study demonstrating the antiproliferative effect of vanadium complexes bearing 8-hydroxyquinoline (quinH) ligands, including the parent and -CH3 (Me), -NO2, -Cl and -I substituted ligands, on HCT116 and A2780 cancer cell lines. To determine the structure-cytotoxicity relationships seven six-coordinate oxovanadium(v) complexes [VO(OMe)(5,7-(Me)2-quin)2] (1), [VO(OMe)(5,7-Cl2-quin)2] (2), [VO(OMe)(5,7-Cl,I-quin)2] (3), [VO(OMe)(5,7-I2-quin)2] (4), [VO(OMe)(5-NO2-quin)2] (5), [VO(OMe)(5-Cl-quin)2] (6), and [VO(OMe)(quin)2] (7) were investigated. The cytotoxicity of 8-hydroxyquinoline oxovanadium(v) complexes is higher in the A2780 cell line (lower IC50) than that observed for the widely used chemotherapeutic agent, cisplatin, while displaying low cytotoxicity for normal human primary fibroblasts. Substituents introduced into the 8-hydroxyquinoline backbone reduced the antiproliferative effect of the vanadium complexes, and the complexes with the ligand substituted only in the 5 position (5 and 6) were more cytotoxic than those with substituents in the 5,7 positions of the quin backbone (1-4). Depending on the substituent type, the cytotoxicity of 1-4 followed the trend: -Cl > -CH3 > -I. Incubation of A2780 cancer cells with IC50 concentrations of complexes 5, 6 and 7 promoted cellular detachment, possibly through membrane destabilization, and triggered apoptosis and necrosis. ROS production might be responsible for the cell death mechanism observed particularly in the A2780 cells exposed to complexes 5 and 6.

Choroba, K, Machura B, Kula S, Raposo LR, Fernandes AR, Kruszynski R, Erfurt K, Shul'pina LS, Kozlov YN, Shul'pin GB.  2019.  Copper(ii) complexes with 2,2':6',2''-terpyridine, 2,6-di(thiazol-2-yl)pyridine and 2,6-di(pyrazin-2-yl)pyridine substituted with quinolines. Synthesis, structure, antiproliferative activity, and catalytic activity in the oxidation of alkanes and alcohols, 2019. Dalton Trans. 48(33):12656-12673. AbstractWebsite

A series of 2,2':6',2''-terpyridine (terpy), 2,6-di(thiazol-2-yl)pyridine (dtpy) and 2,6-di(pyrazin-2-yl)pyridine (dppy) derivatives with n-quinolyl substituents (n = 2 and 4) was used to synthesize five-coordinate complexes [CuCl2(n-quinolyl-terpy)] (1-2), [CuCl2(n-quinolyl-dtpy)] (3-4) and [CuCl2(n-quinolyl-dppy)] (5-6), respectively. The main emphasis of the research was to investigate the impact of the triimine skeleton (terpy, dtpy and dppy) and n-quinolyl pendant substituent on the antiproliferative and catalytic properties of 1-6. The obtained Cu(ii) compounds were studied as antiproliferative agents against human colorectal (HCT116) and ovarian (A2780) carcinoma, and they were used as catalysts for the oxidation of alkanes and alcohols with peroxides under mild conditions. The kinetic characteristics of the oxidizing species generated by the catalytic system Cu(ii) complex-H2O2 in CH3CN were obtained from the dependence of the alkane oxidation rate on its initial concentration. A model of competitive interaction of hydroxyl radicals with CH3CN and RH in the catalyst cavity has been proposed which is based on the simultaneous study of kinetics and selectivity in alkane oxidations.

Choroba, K, Machura B, Raposo LR, Malecki JG, Kula S, Pajak M, Erfurt K, Maron AM, Fernandes AR.  2019.  Platinum(ii) complexes showing high cytotoxicity toward A2780 ovarian carcinoma cells, 2019. Dalton Trans. 48(34):13081-13093. AbstractWebsite

2,6-Bis(thiazol-2-yl)pyridines functionalized with 9-anthryl (L(1)), 9-phenanthryl (L(2)), and 1-pyrenyl (L(3)) groups were used for the preparation of [Pt(L(n))Cl]CF3SO3 (1-3). The constitution of the Pt(ii) complexes was determined by (1)H and (13)C NMR spectroscopy, HR-MS spectrometry, elemental analysis and X-ray analysis (for (1)). The electrochemical and photophysical properties of [Pt(L(n))Cl]CF3SO3 were compared with the behaviour of the Pt(ii) complexes with aryl-substituted 2,2':6',2''-terpyridine ligands. What is noteworthy is that the coordination ability of dtpy toward the Pt(ii) centre was investigated for the first time. All complexes were tested in vitro by MTS assay on four tumor cell lines, A2780 (ovarian carcinoma), HTC116 (colon rectal carcinoma), MCF7 (breast adenocarcinoma), and PC3 (prostate carcinoma) and on normal primary fibroblasts. Compounds (1-3) showed a dose dependent antiproliferative effect in the A2780 cell line with (3) > (2) > (1) and this loss of A2780 cell viability was due to a combination of an apoptotic cell death mechanism via mitochondria and autophagic cell death. Exposure to IC50 concentration of (2) induced an increase in the number of apoptotic nuclei and a depolarization of the mitochondrial membrane which is consistent with the induction of apoptosis while exposure to IC50 concentration of (3) showed an increase in the apoptotic nuclei with a slight hyperpolarization of the mitochondrial membrane that might indicate an initial step of apoptosis induction. The complexes (2) and (3) induce an increase in the production of intracellular ROS which is associated with the trigger of the apoptotic pathways. The ROS production was augmented by the presence of oxidants and correlated with an increase of oxygen radicals. The IC50 of (2) and (3) (4.4 muM and 2.9 muM, respectively) was similar to the IC50 of cisplatin (3.4 muM) in the A2780 cell line, which together with their low cytotoxicity in normal fibroblasts, demonstrates their potential for further studies.

Coimbra, J, Mota C, Santos S, Baptista PV, Fernandes AR.  2015.  Inorganic Compounds Going NANO. Annals of Medicinal Chemistry and Research. 2(1)medicinalchemistry-1-1010.pdf
Conde, J, Larguinho M, Cordeiro A, Raposo LR, Costa PM, Santos S, Diniz MS, Fernandes AR, Baptista PV.  2014.  Gold-nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis. Nanotoxicology. 8(5):521-32.14condenantox.pdf14condenantoxsuppl.pdf
Cordeiro, M, Carvalho L, Silva J, Saúde L, Fernandes AR, Baptista. PV.  2017.  Gold nanobeacons for tracking gene silencing in Zebrafish. Nanomaterials. AbstractWebsite

The use of gold nanoparticles for effective gene silencing has demonstrated its potential as a tool for gene expression experiments and for the treatment of several diseases. Here, we used a gold nanobeacon designed to specifically silence the enhanced green fluorescence protein (EGFP) mRNA in embryos of a fli-EGFP transgenic zebrafish line, while simultaneously allowing the tracking and localization of the silencing events via the beacon’s emission. Fluorescence imaging measurements demonstrated a decrease of the EGFP emission with a concomitant increase in the fluorescence of the Au-nanobeacon. Furthermore, microinjection of the Au-nanobeacon led to a negligible difference in mortality and malformations in comparison to the free oligonucleotide, indicating that this system is a biocompatible platform for the administration of gene silencing moieties. Together, these data illustrate the potential of Au-nanobeacons as tools for in vivo zebrafish gene modulation with low toxicity which may be used towards any gene of interest.

Cordeiro, M, Carlos FF, Pedrosa P, Lopez A, Baptista PV.  2016.  Gold Nanoparticles for Diagnostics: Advances towards Points of Care. Diagnostics. 6(4):43. AbstractWebsite

The remarkable physicochemical properties of gold nanoparticles (AuNPs) have prompted developments in the exploration of biomolecular interactions with AuNP-containing systems, in particular for biomedical applications in diagnostics. These systems show great promise in improving sensitivity, ease of operation and portability. Despite this endeavor, most platforms have yet to reach maturity and make their way into clinics or points of care (POC). Here, we present an overview of emerging and available molecular diagnostics using AuNPs for biomedical sensing that are currently being translated to the clinical setting.

Corvo, L, Mendo AS, Figueiredo S, Larguinho M, Gaspar R, Baptista PV, Fernandes AR.  2016.  Liposomes as delivery system of a Sn(IV) compound for cancer therapy. Pharmaceutical Research. 6(33):1351-8. AbstractWebsite

PROPOSE:
Tin complexes demonstrate antiproliferative activities in some case higher than cisplatin, with IC50 at the low micromolar range. We have previously showed that the cyclic trinuclear complex of Sn(IV) bearing an aromatic oximehydroxamic acid group [nBu2Sn(L)]3 (L=N,2-dihydroxy-5-[N-hydroxyethanimidoyl]benzamide) (MG85) shows high anti-proliferative activity, induces apoptosis and oxidative stress, and causes destabilization of tubulin microtubules, particularly in colorectal carcinoma cells. Despite the great efficacy towards cancer cells, this complex still shows some cytotoxicity to healthy cells. Targeted delivery of this complex specifically towards cancer cells might foster cancer treatment.
METHODS:
MG85 complex was encapsulated into liposomal formulation with and without an active targeting moiety and cancer and healthy cells cytotoxicity was evaluated.
RESULTS:
Encapsulation of MG85 complex in targeting PEGylated liposomes enhanced colorectal carcinoma (HCT116) cancer cell death when compared to free complex, whilst decreasing cytotoxicity in non-tumor cells. Labeling of liposomes with Rhodamine allowed assessing internalization in cells, which showed significant cell uptake after 6 h of incubation. Cetuximab was used as targeting moiety in the PEGylated liposomes that displayed higher internalization rate in HCT116 cells when compared with non-targeted liposomes, which seems to internalize via active binding of Cetuximab to cells.
CONCLUSIONS:
The proposed formulation open new avenues in the design of innovative transition metal-based vectorization systems that may be further extended to other novel metal complexes towards the improvement of their anti-cancer efficacy, which is usually hampered by solubility issues and/or toxicity to healthy tissues.

Czerwinska, K, Machura B, Kula S, Krompiec S, Erfurt K, Roma-Rodrigues C, Fernandes AR, Shul'pina LS, Ikonnikov NS, Shul'pin GB.  2017.  Copper(ii) complexes of functionalized 2,2[prime or minute]:6[prime or minute],2[prime or minute][prime or minute]-terpyridines and 2,6-di(thiazol-2-yl)pyridine: structure, spectroscopy, cytotoxicity and catalytic activity, 2017. Dalton Transactions. 46(29):9591-9604.: The Royal Society of Chemistry AbstractWebsite

Six new copper(ii) complexes with 2,2[prime or minute]:6[prime or minute],2[prime or minute][prime or minute]-terpyridine (4[prime or minute]-Rn-terpy) [1 (R1 = furan-2-yl), 2 (R2 = thiophen-2-yl), and 3 (R3 = 1-methyl-1H-pyrrol-2-yl)] and 2,6-di(thiazol-2-yl)pyridine derivatives (Rn-dtpy) [4 (R1), 5 (R2), and 6 (R3)] have been synthesized by a reaction between copper(ii) chloride and the corresponding ligand. The complexes have been characterized by UV-vis and IR spectroscopy, and their structures have been determined by X-ray analysis. The antiproliferative potential of copper(ii) complexes of 2,2[prime or minute]:6[prime or minute],2[prime or minute][prime or minute]-terpyridine and 2,6-di(thiazol-2-yl)pyridine derivatives towards human colorectal (HCT116) and ovarian (A2780) carcinoma as well as towards lung (A549) and breast adenocarcinoma (MCF7) cell lines was examined. Complex 1 and complex 6 were found to have the highest antiproliferative effect on A2780 ovarian carcinoma cells, particularly when compared with complex 2, 3 with no antiproliferative effect. The order of cytotoxicity in this cell line is 6 > 1 > 5 > 4 > 2 [approximate] 3. Complex 2 seems to be much more specific towards colorectal carcinoma HCT116 and lung adenocarcinoma A549 cells. The viability loss induced by the complexes agrees with Hoechst 33258 staining and typical morphological apoptotic characteristics like chromatin condensation and nuclear fragmentation. The specificity towards different types of cell lines and the low cytotoxic activity towards healthy cells are of particular interest and are a positive feature for further developments. Complexes 1-6 were also tested in the oxidation of alkanes and alcohols with hydrogen peroxide and tert-butyl-hydroperoxide (TBHP). The most active catalyst 4 gave, after 120 min, 0.105 M of cyclohexanol + cyclohexanone after reduction with PPh3. This concentration corresponds to a yield of 23% and TON = 210. Oxidation of cis-1,2-dimethylcyclohexane with m-CPBA catalyzed by 4 in the presence of HNO3 gave a product of a stereoselective reaction (trans/cis = 0.47). Oxidation of secondary alcohols afforded the target ketones in yields up to 98% and TON = 630.