Pedro V. Baptista

Noble Metal Nanoparticles for Biosensing Applications

In the last decade the use of nanomaterials has been having a great impact in biosensing. In particular, the unique properties of noble metal nanoparticles have allowed for the development of new biosensing platforms with enhanced capabilities in the specific detection of bioanalytes. Noble metal nanoparticles show unique physicochemical properties (such as ease of functionalization via simple chemistry and high surface-to-volume ratios) that allied with their unique spectral and optical properties have prompted the development of a plethora of biosensing platforms. Additionally, they also provide an additional or enhanced layer of application for commonly used techniques, such as fluorescence, infrared and Raman spectroscopy. Herein we review the use of noble metal nanoparticles for biosensing strategies—from synthesis and functionalization to integration in molecular diagnostics platforms, with special focus on those that have made their way into the diagnostics laboratory.

Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine

In the last 30 years we have assisted to a massive advance of nanomaterials in material science. Nanomaterials and structures, in addition to their small size, have properties that differ from those of larger bulk materials, making them ideal for a host of novel applications. The spread of nanotechnology in the last years has been due to the improvement of synthesis and characterization methods on the nanoscale, a field rich in new physical phenomena and synthetic opportunities. In fact, the development of functional nanoparticles has progressed exponentially over the past two decades. This work aims to extensively review 30 years of different strategies of surface modification and functionalization of noble metal (gold) nanoparticles, magnetic nanocrystals and semiconductor nanoparticles, such as quantum dots. The aim of this review is not only to provide in-depth insights into the different biofunctionalization and characterization methods, but also to give an overview of possibilities and limitations of the available nanoparticles.

Noble Metal Nanoparticles Applications in Cancer

Nanotechnology has prompted new and improved materials for biomedical applications with particular emphasis in therapy and diagnostics. Special interest has been directed at providing enhanced molecular therapeutics for cancer, where conventional approaches do not effectively differentiate between cancerous and normal cells; that is, they lack specificity. This normally causes systemic toxicity and severe and adverse side effects with concomitant loss of quality of life. Because of their small size, nanoparticles can readily interact with biomolecules both at surface and inside cells, yielding better signals and target specificity for diagnostics and therapeutics. This way, a variety of nanoparticles with the possibility of diversified modification with biomolecules have been investigated for biomedical applications including their use in highly sensitive imaging assays, thermal ablation, and radiotherapy enhancement as well as drug and gene delivery and silencing. Here, we review the available noble metal nanoparticles for cancer therapy, with particular focus on those already being translated into clinical settings.

Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

Gold Nanoparticles for Diagnostics: Advances towards Points of Care

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.

Gold and silver nanoparticles for clinical diagnostics - From genomics to proteomics.

Nanotechnology has prompted researchers to develop new and improved materials aimed at biomedical applications with particular emphasis in diagnostics and therapy. Special interest has been directed at providing enhanced biomolecular diagnostics, including SNP detection gene expression profiles and biomarker characterisation. These strategies have focused on the development of nanoscale devices and platforms that can be used for single molecule characterisation of nucleic acid, DNA or RNA, and protein at an increased rate when compared to traditional techniques. Also, several advances have been reported on DNA analysis in real time, at both high resolution and very high throughputs, suitable for biomedical diagnostics. Here, we shall provide a review of available nanotechnology-based platforms for biomolecular recognition, and their application to molecular diagnostics and genome analysis, with emphasis on the use of noble metal nanoparticles for simple and specific analysis systems. Particular focus will be put on those already being translated into clinical settings. This article is part of a Special Issue entitled: Proteomics: The clinical link.

In vivo tumor targeting via nanoparticle-mediated therapeutic siRNA coupled to inflammatory response in lung cancer mouse models

Up to now, functionalized gold nanoparticles have been optimized as an effective intracellular in vitro delivery vehicle for siRNAs to interfere with the expression of specific genes by selective targeting, and provide protection against nucleases. Few examples however of suchlike in vivo applications have been described so far. In this study, we report the use of siRNA/RGD gold nanoparticles capable of targeting tumor cells in a lung cancer syngeneic orthotopic murine model. Therapeutic RGD-nanoparticle treatment resulted in successful targeting evident from significant c-myc oncogene down-regulation followed by tumor growth inhibition and prolonged survival of lung tumor bearing mice, possibly via αvβ3 integrin interaction. Our results suggest that RGD gold nanoparticles-mediated delivery of siRNA by intratracheal instillation in mice leads to successful suppression of tumor cell proliferation and respective tumor size reduction. These results reiterate the capability of functionalized gold nanoparticles for targeted delivery of siRNA to cancer cells towards effective silencing of the specific target oncogene. What is more, we demonstrate that the gold-nanoconjugates trigger a complex inflammatory and immune response that might promote the therapeutic effect of the RNAi to reduce tumor size with low doses of siRNA.

A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper.

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.

Heterocyclic Anticancer Compounds: Recent Advances and the Paradigm Shift towards the Use of Nanomedicine’s Tool Box

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.

Exosome in Tumour Microenvironment: Overview of the Crosstalk between Normal and Cancer Cells

Cancer development is a multistep process in which exosomes play important roles. Exosomes are small vesicles formed in vesicular bodies in the endosomal network. The major role of exosomes seems to be the transport of bioactive molecules between cells. Depending on the cell of origin, exosomes are implicated in the regulation of several cellular events, with phenotypic consequences in recipient cells. Cancer derived exosomes (CCEs) are important players in the formation of the tumour microenvironment by (i) enabling the escape of tumour cells to immunological system and help initiating the inflammatory response; (ii) acting in the differentiation of fibroblasts and mesenchymal cells into myofibroblasts; (iii) triggering the angiogenic process; and (iv) enhancing the metastatic evolution of the tumour by promoting epithelial to mesenchymal transformation of tumour cells and by preparing the tumour niche in the new anatomical location. Since the finding that exosomes content resembles that of the cell of origin, they may be regarded as suitable biomarkers for cancer diagnosis, allowing for diagnosis and prognosis via a minimal invasive procedure. Exosome involvement in cancer may open new avenues regarding therapeutics, such as vectors for targeted drug delivery.