Publications

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2018
Evaluation of cell toxicity and DNA and protein binding of green synthesized silver nanoparticles, Ribeiro, A. P. C., Anbu S., Alegria E. C. B. A., Fernandes A. R., Baptista P. V., Mendes R., Matias A. S., Mendes M., Guedes da Silva M. F. C., and Pombeiro A. J. L. , Biomedicine & Pharmacotherapy, 2018, Volume 101, p.137 - 144, (2018) AbstractWebsite

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Multifunctional microfluidic chip for optical nanoprobe based RNA detection – application to Chronic Myeloid Leukemia, Alves, Pedro Urbano, Vinhas Raquel, Fernandes Alexandra R., Birol Semra Zuhal, Trabzon Levent, Bernacka-Wojcik Iwona, Igreja Rui, Lopes Paulo, Baptista Pedro Viana, Águas Hugo, Fortunato Elvira, and Martins Rodrigo , Scientific Reports, 2018, Volume 8, Issue 1, p.381, (2018) 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.

Optical and Structural Characterization of a Chronic Myeloid Leukemia DNA Biosensor, Cordeiro, Mílton, Otrelo-Cardoso Ana Rita, Svergun Dmitri I., Konarev Petr V., Lima João Carlos, Santos-Silva Teresa, and Baptista Pedro Viana , ACS Chemical BiologyACS Chemical Biology, 2018, Volume 13, Issue 5, p.1235 - 1242, (2018) AbstractWebsite

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Synthesis, Cytotoxicity Evaluation in Human Cell Lines and in Vitro DNA Interaction of a Hetero‐Arylidene‐9(10H)‐Anthrone, Daniela, Peixoto, Margarida Figueiredo, Gabriela Malta, Catarina Roma‐Rodrigues, V. Baptista Pedro, R. Fernandes Alexandra, Sónia Barroso, Luísa Carvalho Ana, M. Afonso Carlos A., M. Ferreira Luisa, and S. Branco Paula , European Journal of Organic ChemistryEuropean Journal of Organic Chemistry, 2018, Volume 2018, Issue 4, p.545 - 549, (2018) AbstractWebsite

A new and never before reported hetero?arylidene?9(10H)?anthrone structure (4) was unexpectedly isolated on reaction of 1,2?dimethyl?3?ethylimidazolium iodide (2) and 9?anthracenecarboxaldehyde (3) under basic conditions. Its structure was unequivocally confirmed by X?ray crystallography. No cytotoxicity in human healthy fibroblasts and in two different cancer cell lines was observed, indicating its applicability in biological systems. Compound 4 interacts with CT?DNA by intercalation between the adjacent base pairs of DNA with a high binding affinity [Kb = 2.0?(±0.20)???105 m?1], which is 10?? higher than that described for doxorubicin [Kb = 3.2?(±0.23)???104 m?1]. Furthermore, compound 4 quenches the fluorescence emission of a GelRed?CT?DNA system with a quenching constant (KSV) of 3.3?(±0.3)???103 m?1 calculated by the Stern?Volmer equation.

Molecular Diagnostics of Chronic Myeloid Leukemia - precision medicine via gold nanoparticles, Vinhas, Raquel, Fernandes Alexandra R., and Baptista Pedro V. , In CDx and precision medicine for hematologic malignancies, p.205-230, (2018)
A novel BCR-ABL1 mutation in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia, Vinhas, Raquel, Lourenço Alexandra, Santos Susana, Lemos Marcos, Ribeiro Patrícia, de Sousa Aida Botelho, Baptista Pedro V., and Fernandes Alexandra R. , p.8589—8598, (2018)
Combination of chemotherapy and Au-nanoparticle photothermy in the visible light to tackle doxorubicin resistance in cancer cells, Pedrosa, Pedro, Mendes Rita, Cabral Rita, Martins Luisa M. D. R. S., Baptista Pedro V., and Fernandes Alexandra R. , Scientific Reports, Volume 8, (2018) Abstract

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A double Philadelphia chromosome-positive chronic myeloid leukemia patient, co-expressing P210(BCR-ABL1) and P195(BCR-ABL1) isoforms, Vinhas, Raquel, Lourenco Alexandra, Santos Susana, Ribeiro Patricia, Silva Madalena, de Sousa Aida Botelho, Baptista Pedro V., and Fernandes Alexandra R. , Haematologica, Volume 103, Number 11, p.E549-E552, (2018) Abstract

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Gold nanoprobe-based non-crosslinking hybridization for molecular diagnostics: an update, Baptista, P. V. , Expert Rev Mol Diagn, Volume 18, Number 9, p.767-773, (2018) AbstractWebsite

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The Important Role of the Nuclearity, Rigidity, and Solubility of Phosphane Ligands in the Biological Activity of Gold(I) Complexes, Svahn, Noora, Moro Artur J., Roma-Rodrigues Catarina, Puttreddy Rakesh, Rissanen Kari, Baptista Pedro V., Fernandes Alexandra R., Lima João Carlos, and Rodríguez Laura , Chemistry – A European Journal, Volume 24, Number 55, p.14654-14667, (2018) AbstractWebsite

Abstract A series of 4-ethynylaniline gold(I) complexes containing monophosphane (1,3,5-triaza-7-phosphaadamantane (pta; 2), 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (3), and PR3, with R=naphthyl (4), phenyl (5), and ethyl (6)) and diphosphane (bis(diphenylphosphino)acetylene (dppa; 7), trans-1,2-bis(diphenylphosphino)ethene (dppet; 8), 1,2-bis(diphenylphosphino)ethane (dppe; 9), and 1,3-bis(diphenylphosphino)propane (dppp; 10)) ligands have been synthesized and their efficiency against tumor cells evaluated. The cytotoxicity of complexes 2–10 was evaluated in human colorectal (HCT116) and ovarian (A2780) carcinoma as well as in normal human fibroblasts. All the complexes showed a higher antiproliferative effect in A2780 cells, with the cytotoxicity decreasing in the following order 5>6=9=10>8>2>4>7>3. Complex 4 stands out for its very high selectivity towards ovarian carcinoma cells (IC50=2.3 μm) compared with colorectal carcinoma and normal human fibroblasts (IC50>100 μm), which makes this complex very attractive for ovarian cancer therapy. Its cytotoxicity in these cells correlates with the induction of the apoptotic process and an increase of intracellular reactive oxygen species (ROS). The effects of the nuclearity, rigidity, and solubility of these complexes on their biological activity were also analyzed. X-ray crystal structure determination allowed the identification of short N−H⋅⋅⋅π contacts as the main driving forces for the three-dimensional packing in these molecules.

Nano-Strategies to Fight Multidrug Resistant Bacteria—“A Battle of the Titans”, Baptista, Pedro V., McCusker Matthew P., Carvalho Andreia, Ferreira Daniela A., Mohan Niamh M., Martins Marta, and Fernandes Alexandra R. , Frontiers in Microbiology, Volume 9, p.1441, (2018) 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.

Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells, McCully, Mark, Conde Joao, Baptista Pedro V., Mullin Margaret, Dalby Matthew J., and Berry Catherine C. , Plos One, Volume 13, Number 2, (2018) Abstract

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POxylated Dendrimer-Based Nano-in-Micro Dry Powder Formulations for Inhalation Chemotherapy, Restani, Rita B., Pires Rita F., Tolmatcheva Anna, Cabral Rita, Baptista, Pedro V., Fernandes Alexandra R., Casimiro Teresa, Bonifacio Vasco D. B., and Aguiar-Ricardo Ana , Chemistryopen, Volume 7, Number 10, p.772-779, (2018) Abstract

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2017
Nanoparticles—Emerging Potential for Managing Leukemia and Lymphoma, Vinhas, Raquel, Mendes Rita, Fernandes Alexandra R., and Baptista Pedro V. , Front. Bioeng. Biotechnol, 2017, Volume 5, p.79, (2017) AbstractWebsite

Nanotechnology has become a powerful approach to improve the way we diagnose and treat cancer. In particular, nanoparticles possess unique features for enhanced sensitivity and selectivity for earlier detection of circulating cancer biomarkers. In vivo, nanoparticles enhance the therapeutic efficacy of anticancer agents when compared to conventional chemotherapy, improving vectorization and delivery, and helping to overcome drug resistance. Nanomedicine has been mostly focused on solid cancers due to take advantage from the enhanced permeability and retention (EPR) effect experienced by tissues in the close vicinity of tumors, which enhance nanomedicine’s accumulation and, consequently, improve efficacy. Nanomedicines for leukemia and lymphoma, where EPR effect is not a factor, are addressed differently from solid tumors. Nevertheless, nanoparticles have provided innovative approaches to simple and non-invasive methodologies for diagnosis and treatment in liquid tumors. In this review, we consider the state of the art on different types of nanoconstructs for the management of liquid tumors, from pre-clinical studies to clinical trials. We also discuss the advantages of nanoplatforms for theranostics and the central role played by nanoparticles in this combined strategy.

Gold nanobeacons for tracking gene silencing in Zebrafish, Cordeiro, Milton, Carvalho Lara, Silva Joana, Saúde Leonor, Fernandes Alexandra R., and Baptista. Pedro V. , Nanomaterials, (2017) 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.

Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer—The Case for Combined Delivery?, Mendes, Rita, Fernandes Alexandra R., and Baptista Pedro V. , Genes, Volume 3, Issue 8, p.94, (2017) AbstractWebsite

Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA) sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs) have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics

Gold Nanoparticles for BCR-ABL1 Gene Silencing: Improving Tyrosine Kinase Inhibitor Efficacy in Chronic Myeloid Leukemia, Vinhas, Raquel, Fernandes Alexandra, and Baptista Pedro V. , Molecular Therapy Nucleic Acids, Volume 7, p.408-416, (2017) AbstractWebsite

Introduction of tyrosine kinase inhibitors for chronic myeloid leukemia treatment is associated with a 63% probability of maintaining a complete cytogenetic response, meaning that over 30% patients require an alternative methodology to overcome resistance, tolerance, or side effects. Considering the potential of nanotechnology in cancer treatment and the benefits of a combined therapy with imatinib, a nanoconjugate was designed to achieve BCR-ABL1 gene silencing. Gold nanoparticles were functionalized with a single-stranded DNA oligonucleotide that selectively targets the e14a2 BCR-ABL1 transcript expressed by K562 cells. This gold (Au)-nanoconjugate showed great efficacy in gene silencing that induced a significant increase in cell death. Variation of BCL-2 and BAX protein expression, an increase of caspase-3 activity, and apoptotic bodies in cells treated with the nanoconjugate demonstrate its aptitude for inducing apoptosis on K562 BCR-ABL1-expressing cells. Moreover, the combination of the silencing Au-nanoconjugate with imatinib prompted a decrease of imatinib IC50. This Au-nanoconjugate was also capable of inducing the loss of viability of imatinib-resistant K562 cells. This strategy shows that combination of Au-nanoconjugate and imatinib make K562 cells more vulnerable to chemotherapy and that the Au-nanoconjugate alone may overcome imatinib-resistance mechanisms, thus providing an effective treatment for chronic myeloid leukemia patients who exhibit drug tolerance.

Tumor microenvironment modulation via gold nanoparticles targeting malicious exosomes: implications in cancer diagnostics and Therapy, Roma-Rodrigues, Catarina, Raposo Luis, Cabral Rita, Paradinha Fabiana, Baptista Pedro V., and Fernandes Alexandra R. , Int. J. Mol. Sci., Volume 18, Issue 1, p.162, (2017) AbstractWebsite

Exosomes are nanovesicles formed in the endosomal pathway with an important role in paracrine and autocrine cell communication. Exosomes secreted by cancer cells, malicious exosomes, have important roles in tumor microenvironment maturation and cancer progression. The knowledge of the role of exosomes in tumorigenesis prompted a new era in cancer diagnostics and therapy, taking advantage of the use of circulating exosomes as tumor biomarkers due to their stability in body fluids and targeting malignant exosomes’ release and/or uptake to inhibit or delay tumor development. In recent years, nanotechnology has paved the way for the development of a plethora of new diagnostic and therapeutic platforms, fostering theranostics. The unique physical and chemical properties of gold nanoparticles (AuNPs) make them suitable vehicles to pursuit this goal. AuNPs’ properties such as ease of synthesis with the desired shape and size, high surface:volume ratio, and the possibility of engineering their surface as desired, potentiate AuNPs’ role in nanotheranostics, allowing the use of the same formulation for exosome detection and restraining the effect of malicious exosomes in cancer progression.

Allele specific LAMP- gold nanoparticle for characterization of single nucleotide polymorphisms, Carlos, F. F., Veigas B., Matias A. S., Doria G., Flores O., and Baptista P. V. , Biotechnol Rep (Amst), Volume 16, p.21-25, (2017) AbstractWebsite

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Digital Microfluidics for Nucleic Acid Amplification, Coelho, Beatriz, Veigas Bruno, Fortunato Elvira, Martins Rodrigo, Aguas Hugo, Igreja Rui, and Baptista Pedro V. , Sensors, Volume 17, Number 7, (2017) Abstract

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A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification Detection, Coelho, Beatriz Jorge, Veigas Bruno, Aguas Hugo, Fortunato Elvira, Martins Rodrigo, Baptista Pedro Viana, and Igreja Rui , Sensors, Volume 17, Number 11, (2017) Abstract

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Gene Silencing Using Multifunctionalized Gold Nanoparticles for Cancer Therapy., Fernandes, Alexandra R., and Baptista Pedro V. , Methods in molecular biology (Clifton, N.J.), Volume 1530, p.319-336, (2017) Abstract

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Immortalization and characterization of a new canine mammary tumour cell line FR37-CMT, Raposo, L. R., Roma-Rodrigues C., Faisca P., Alves M., Henriques J., Carvalheiro MC, Corvo M. L., Baptista P. V., Pombeiro A. J., and Fernandes A. R. , Veterinary and Comparative Oncology, Volume 15, Number 3, p.952-967, (2017) Abstract

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Multifunctional gold-nanoparticles: A nanovectorization tool for the targeted delivery of novel chemotherapeutic agents, Fernandes, Alexandra R., Jesus Joao, Martins Pedro, Figueiredo Sara, Rosa Daniela, Martins Luisa M. R. D. R. S., Corvo Maria Luisa, Carvalheiro Manuela C., Costa Pedro M., and Baptista Pedro V. , Journal of Controlled Release, Volume 245, p.52-61, (2017) Abstract

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Nanoparticles-Emerging Potential for Managing Leukemia and Lymphoma, Vinhas, Raquel, Mendes Rita, Fernandes Alexandra R., and Baptista Pedro V. , Frontiers in Bioengineering and Biotechnology, Volume 5, (2017) Abstract

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