Publications

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2015
Gold nanoparticle-siRNA mediated oncogene knockdown at RNA and protein level, with associated gene effects, Child, Hannah Winifred, Hernandez Yulan, Conde João, Mullin Margaret, Baptista Pedro V., de la Fuente Jesus Maria, and Berry Catherine C. , NANOMEDICINE, Volume 10, Issue 16, p.2513-2525, (2015) AbstractWebsite

Aims: RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells. Results: Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses. Discussion: The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes. Conclusion: The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels.

Gold Nanoparticles for DNA/RNA-Based Diagnostics, Franco, Ricardo, Pedrosa Pedro, Carlos Fábio Ferreira, Veigas Bruno, and Baptista Pedro Viana , Handbook of Nanoparticles, Berlin, p.1-25, (2015) Abstract

The remarkable physicochemical properties of gold nanoparticles (AuNPs) have prompted development
in exploring biomolecular interactions with AuNPs-containing systems, pursuing biomedical applications
in diagnostics. Among these applications, AuNPs have been remarkably useful for the development of
DNA/RNA detection and characterization systems for diagnostics, including systems suitable for point of
need. Here, emphasis will be on available molecular detection schemes of relevant pathogens and their
molecular characterization, genomic sequences associated with medical conditions (including cancer),
mutation and polymorphism identification, and the quantification of gene expression.

Gold Nanotheranostics: Proof-of-Concept or Clinical Tool?, Pedrosa, Pedro, Vinhas Raquel, Fernandes Alexandra, and Baptista Pedro V. , Nanomaterials, Volume 5, Issue 4, p.1853-1879, (2015) AbstractWebsite

Nanoparticles have been making their way in biomedical applications and personalized medicine, allowing for the coupling of diagnostics and therapeutics into a single nanomaterial—nanotheranostics. Gold nanoparticles, in particular, have unique features that make them excellent nanomaterials for theranostics, enabling the integration of targeting, imaging and therapeutics in a single platform, with proven applicability in the management of heterogeneous diseases, such as cancer. In this review, we focus on gold nanoparticle-based theranostics at the lab bench, through pre-clinical and clinical stages. With few products facing clinical trials, much remains to be done to effectively assess the real benefits of nanotheranostics at the clinical level. Hence, we also discuss the efforts currently being made to translate nanotheranostics into the market, as well as their commercial impact.

Heterocyclic Anticancer Compounds: Recent Advances and the Paradigm Shift towards the Use of Nanomedicine’s Tool Box, Martins, Pedro, Jesus João, Santos Sofia, Raposo Luis R., Roma-Rodrigues Catarina, Baptista Pedro Viana, and Fernandes Alexandra , Molecules, Issue 20, p.16852-16891, (2015) AbstractWebsite

The majority of heterocycle compounds and typically common heterocycle fragments present in most pharmaceuticals currently marketed, alongside with their intrinsic versatility and unique physicochemical properties, have poised them as true cornerstones of medicinal chemistry. Apart from the already marketed drugs, there are many other being investigated for their promising activity against several malignancies. In particular, anticancer research has been capitalizing on the intrinsic versatility and dynamic core scaffold of these compounds. Nevertheless, as for any other promising anticancer drugs, heterocyclic compounds do not come without shortcomings. In this review, we provide for a concise overview of heterocyclic active compounds and families and their main applications in medicine. We shall focus on those suitable for cancer therapy while simultaneously addressing main biochemical modes of action, biological targets, structure-activity relationships as well as intrinsic limitation issues in the use of these compounds. Finally, considering the advent of nanotechnology for effective selective targeting of drugs, we shall discuss fundamental aspects and considerations on nanovectorization of such compounds that may improve pharmacokinetic/pharmacodynamic properties of heterocycles.

Nanoparticles for Diagnostics and Imaging, Larguinho, Miguel, Figueiredo Sara, Cordeiro Ana, Carlos Fábio Ferreira, Cordeiro Milton, Pedrosa Pedro, and Baptista Pedro Viana , Frontiers in Nanomedicine, p.3-46, (2015) Abstractsample.pdf

Nanoparticles possess unique optical and physic-chemical properties that may potentiate applications in biomedicine, in particular in diagnostics, therapy and imaging. Advances on biomolecular diagnostics strategies have greatly focused on single molecule detection and characterization of DNA, RNA or proteins through improved nanoparticle-based platforms. Nanoparticles improve analytical capability when compared to traditional techniques with high resolution and medium-high throughput. Also, particular interest has been directed at SNP detection, gene expression profiles and biomarker characterization through colorimetric, spectrometric or electrochemical strategies.
Molecular imaging has also benefited from the introduction of nanoparticles in standard techniques towards non-invasive imaging procedures that can be used to highlight regions of interest, allowing the characterization of biological processes at the cellular and/or molecular level. Several imaging modalities are associated with low sensitivity, an issue that can be tackled by the use of probes, e.g. contrast agents for X-ray and magnetic resonance imaging, radiolabelled molecules for nuclear medicine. Furthermore, nanoparticles can be used as vehicles that deliver specifically these contrast agents, leading to overcome the limitations of conventional modalities.
This chapter will discuss the use of nanoparticles in biomolecular recognition and imaging applications, focusing those already being translated into clinical settings. Current knowledge will be addressed as well as its evolution towards the future of nanoparticle-based biomedical applications.

Organometallic Compounds in Cancer Therapy: Past Lessons and Future Directions, Martins, Pedro, Marques Mara, Coito Lidia, Pombeiro Armando, Baptista Pedro V., and Fernandes Alexandra R. , ANTI-CANCER AGENTS IN MEDICINAL CHEMISTRY, Volume 9, Issue 14, (2015) AbstractWebsite

Over the past few years, modern medicinal chemistry has evolved towards providing us new and alternative chemotherapeutic compounds with high cytotoxicity towards tumor cells, alongside with reduced side effects in cancer patients. Organometallic compounds and their unique physic-chemical properties typically used in homogenous catalysis are now being translated as potential candidates for medical purposes. Their structural diversity, ligand exchange, redox and catalytic properties make them promising drug candidates for cancer therapy. Over the last decade this area has witnessed a steady growth and a few organometallic compounds have in fact already entered clinical trials, emphasizing its increasing importance and clinical relevance. Here we intend to stress out the different applications of organometallic compounds in medicine with emphasis on cancer therapy, as well as address setbacks regarding formulation issues, systemic toxicity and off-target effects. Advantages over classical coordination metal complexes, their nanovectorisation and specific molecular targets are also discussed.

POxylated Polyurea Dendrimers: Smart Core-Shell Vectors with IC50 Lowering Capacity, Restani, Rita B., Conde Joao, Pires Rita F., Martins Pedro, Fernandes Alexandra R., Baptista Pedro V., and Bonifacio Vasco D. B. , MACROMOLECULAR BIOSCIENCE, Volume 15, Issue 8, p.1045–1051, (2015) AbstractWebsite

The design and preparation of highly efficient drug delivery platforms using green methodologies is at the forefront of nanotherapeutics research. POxylated polyurea dendrimers are efficiently synthesized using a supercritical-assisted polymerization in carbon dioxide. These fluorescent, pH-responsive and water-soluble core-shell smart nanocarriers show low toxicity in terms of cell viability and absence of glutathione depletion, two of the major side effect limitations of current vectors. The materials are also found to act as good transfection agents, through a mechanism involving an endosomal pathway, being able to reduce 100-fold the IC50 of paclitaxel.

The significance of the balance between intracellular glutathione and polyethylene glycol (PEG) for successful siRNA release from gold nanoparticles, McCully, Mark, Hernandez Yulan, Conde João, Baptista Pedro V., de la Fuente Jesus M., Hursthouse Andrew, and St David , Nano Research, (2015) AbstractWebsite

The therapeutic promise of small interfering RNAs (siRNAs) for specific gene silencing is dependent on the
successful delivery of functional siRNAs to the cell cytoplasm. Their conjugation to an established delivery
platform, such as gold nanoparticles, offers a huge potential for treating diseases and advancing our
understanding of cellular processes. The success or failure is dependent on both the uptake of the nanoparticlesinto the cells and subsequent intracellular release of the functional siRNA. In this paper, utilising gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol (PEG) ligand density on knockdown, with a view to determine the optimal nanoparticle
design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, and also the possible effect of steric hindrance in terms of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, whilst knockdown was determined at the protein and physiological levels (cells in S-phase) by in-cell westerns and BrdU incorporation, respectively.

DNA adduct identification using gold-aptamer nanoprobes, Larguinho, Miguel, Santos Sofia, Almeida Joao, and Baptista Pedro V. , Iet Nanobiotechnology, Volume 9, Number 2, p.95-101, (2015) Abstract

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Field Effect Sensors for Nucleic Acid Detection: Recent Advances and Future Perspectives, Veigas, Bruno, Fortunato Elvira, and Baptista Pedro V. , Sensors, Volume 15, Number 5, p.10380-10398, (2015) Abstract

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Gold nanoprobe-based non-crosslinking hybridization for molecular diagnostics, Larguinho, Miguel, Canto Rafaela, Cordeiro Milton, Pedrosa Pedro, Fortuna Andreia, Vinhas Raquel, and Baptista Pedro V. , Expert Review of Molecular Diagnostics, Volume 15, Number 10, p.1355-1368, (2015) Abstract

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GOLD NANOPROBES IN THE DIAGNOSTIC OF CHRONIC MYELOID LEUKEMIA: DETECTION OF THE E14A2 BCR-ABL TRANSCRIPT DIRECTLY IN RNA SAMPLES, Vinhas, R., Correia C., Ribeiro P., Lourenco A., Sousa A., Fernandes A., and Baptista P. , Leukemia Research, Volume 39, p.S90, (2015) Abstract

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Mobile based gold nanoprobe TB diagnostics for point-of-need., Veigas, B., Fortunato E., and Baptista P. V. , Methods in molecular biology (Clifton, N.J.), Volume 1256, p.41-56, (2015) Abstract

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One nanoprobe, two pathogens: gold nanoprobes multiplexing for point-of-care, Veigas, Bruno, Pedrosa Pedro, Carlos Fabio F., Mancio-Silva Liliana, Grosso Ana Rita, Fortunato Elvira, Mota Maria M., and Baptista Pedro V. , Journal of Nanobiotechnology, Volume 13, (2015) Abstract

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RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing, Conde, Joao, Tian Furong, Hernandez Yulan, Bao Chenchen, Baptista Pedro V., Cui Daxiang, Stoeger Tobias, and de la Fuente Jesus M. , Nanoscale, Volume 7, Number 19, p.9083-9091, (2015) Abstract

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Scalable approach for the production of functional DNA based gold nanoprobes, Veigas, Bruno, Portugal Carla, Valerio Rita, Fortunato Elvira, Crespo Joao G., and Baptista Pedro V. , Journal of Membrane Science, Volume 492, p.528-535, (2015) Abstract

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Significance of the balance between intracellular glutathione and polyethylene glycol for successful release of small interfering RNA from gold nanoparticles, McCully, Mark, Hernandez Yulan, Conde Joao, Baptista Pedro V., de la Fuente Jesus M., Hursthouse Andrew, Stirling David, and Berry Catherine C. , Nano Research, Volume 8, Number 10, p.3281-3292, (2015) Abstract

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Single Nucleotide Polymorphism Detection Using Gold Nanoprobes and Bio-Microfluidic Platform With Embedded Micro lenses, Bernacka-Wojcik, Iwona, Aguas Hugo, Carlos Fabio Ferreira, Lopes Paulo, Wojcik Pawel Jerzy, Costa Mafalda Nascimento, Veigas Bruno, Igreja Rui, Fortunato Elvira, Baptista Pedro Viana, and Martins Rodrigo , Biotechnology and Bioengineering, Volume 112, Number 6, p.1210-1219, (2015) Abstract

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2014
Experimental optimization of a passive planar rhombic micromixer with obstacles for effective mixing in a short channel length, Bernacka-Wojcik, Iwona, Ribeiro Susana, Wojcik Pawel Jerzy, Alves Pedro Urbano, Busani Tito, Fortunato Elvira, Baptista Pedro Viana, Covas José António, Águas Hugo, Hilliou Loic, and Martins Rodrigo , RSC ADVANCES, Volume 4, Issue 99, (2014) AbstractWebsite

This paper presents the performance of a passive planar rhombic micromixer with diamond-shaped obstacles and a rectangular contraction between the rhombi. The device was experimentally optimized using water for high mixing efficiency and a low pressure drop over a wide range of Reynolds numbers (Re = 0.1–117.6) by varying geometrical parameters such as the number of rhombi, the distance between obstacles and the contraction width. Due to the large amount of data generated, statistical methods were used to facilitate and improve the results of the analysis. The results revealed a rank of factors influencing mixing efficiency: Reynolds number > number of rhombi > contraction width > inter-obstacles distance. The pressure drop measured after three rhombi depends mainly on Re and inter-obstacle distance. The resulting optimum geometry for the low Re regime has a contraction width of 101 μm and inter-obstacles distance of 93 μm, while for the high Re regime a contraction width of 400 μm and inter-obstacle distance of 121 μm are more appropriate. These mixers enabled 80% mixing efficiency creating a pressure drop of 6.0 Pa at Re = 0.1 and 5.1 × 104 Pa at Re = 117.6, with a mixer length of 2.5 mm. To the authors' knowledge, the developed mixer is one of the shortest planar passive micromixers reported to date.

Characterization of genomic SNP via colorimetric detection using a single gold nanoprobe, Carlos, Fábio Ferreira, Flores Orfeu, Doria Gonçalo, and Baptista Pedro Viana , Analytical Biochemistry, Volume 465, p.1-5, (2014) AbstractWebsite

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au-nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au-nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au-nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey's test to ascertain whether there were any differences between Au-nanoprobe genotyped groups. For the first time, we show that the use of a single Au-nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).

Gold nanobeacons: A potential nanotheranostics platform, Baptista, Pedro Viana , Nanomedicine, Volume 9, Issue 15, p.2247-50, (2014) Website
Gold nanoprobes for multi loci assessment of multi-drug resistant tuberculosis, Pedrosa, Pedro, Veigas Bruno, Machado Diana, Couto Isabel, Viveiros Miguel, and Baptista Pedro V. , Tuberculosis, Volume 94, Issue 3, p.332-337, (2014) AbstractWebsite

Tuberculosis, still one of the leading human infectious diseases, reported 8.7 million new cases in 2011 alone. Also, the increasing rate of multidrug-resistant tuberculosis (MDRTB) and its treatment difficulties pose a serious public health threat especially in developing countries. Resistance to isoniazid and rifampicin, first line antibiotics, is commonly associated with point mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis complex (MTBC). Therefore, the development of cheap, fast and simple molecular methods to assess susceptibility profiles would have a huge impact in the capacity of early diagnosis and treatment of MDRTB.

Gold nanoparticles functionalized with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for MTBC and single base mutations associated with antibiotic resistance, namely the characterization of the three most relevant codons in rpoB gene associated to rifampicin resistance. Here we extend the Au-nanoprobe approach towards discriminating specific mutations within inhA and rpoB genes in PCR amplified DNA from isolates. Using a multiplex PCR reaction for these two genes, it is possible to assess both loci in parallel, and extend the potential of the Au-nanoprobe method to MDRTB molecular characterization with special application in the most frequent Portuguese genotypes.

Gold-Nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis, Conde, João, Larguinho Miguel, Cordeiro Ana, Raposo Luis R., Costa Pedro M., Santos Susana, Diniz Mário, Fernandes Alexandra R., and Baptista Pedro Viana , Nanotoxicology, Volume 8, Issue 5, p.521-532, (2014) AbstractWebsite

Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3′-Cy3 and 5′-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.

Histopathological findings on Carassius auratus hepatopancreas upon exposure to acrylamide: correlation with genotoxicity and metabolic alterations., Larguinho, Miguel, Costa Pedro M., Sousa Gonçalo, Costa Maria H., Diniz Mário S., and Baptista Pedro V. , Journal of Applied Toxicology , Volume 34, Issue 12, (2014)
Low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing - Lab-on-Paper, Costa, Mafalda, Veigas Bruno, Jacob Jorge, Santos David, Gomes Jacinto, Baptista Pedro V., Martins Rodrigo, Inácio João, and Fortunato Elvira , NANOTECHNOLOGY, Volume 9, Issue 25, p.094006, (2014) AbstractWebsite

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.