A comparison of gold nanoparticle surface co-functionalization approaches using Polyethylene Glycol (PEG) and the effect on stability, non-specific protein adsorption and internalization

To create clinically useful gold nanoparticle (AuNP) based cancer therapeutics it is necessary to co-functionalize the AuNP surface with a range of moieties; e.g. Polyethylene Glycol (PEG), peptides and drugs. AuNPs can be functionalized by creating either a mixed monolayer by attaching all the moieties directly to the surface using thiol chemistry, or by binding groups to the surface by means of a bifunctional polyethylene glycol (PEG) linker. The linker methodology has the potential to enhance bioavailability and the amount of functional agent that can be attached. While there is a large body of published work using both surface arrangements independently, the impact of attachment methodology on stability, non-specific protein adsorption and cellular uptake is not well understood, with no published studies directly comparing the two most frequently employed approaches. This paper compares the two methodologies by synthesizing and characterizing PEG and Receptor Mediated Endocytosis (RME) peptide co-functionalized AuNPs prepared using both the mixed monolayer and linker approaches. Successful attachment of both PEG and RME peptide using the two methods was confirmed using Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy and gel electrophoresis. It was observed that while the 'as synthesized' citrate capped AuNPs agglomerated under physiological salt conditions, all the mixed monolayer and PEG linker capped samples remained stable at 1M NaCl, and were stable in PBS over extended periods. While it was noted that both functionalization methods inhibited non-specific protein attachment, the mixed monolayer samples did show some changes in gel electrophoresis migration profile after incubation with fetal calf serum. PEG renders the AuNP stable in-vivo however, studies with MDA-MB-231 and MCF 10A cell lines indicated that functionalization with PEG, blocks cellular uptake. It was observed that co-functionalization with RME peptide using both the mixed monolayer and PEG linker methods greatly enhanced cellular internalization compared to PEG capped AuNPs.

Phosphatidylethanolamines glycoxidation: products and significance

As fosfatidiletanolaminas constituem a segunda classe de fosfolípidos mais abundantes nos organismos. Elas estão presentes nas membranas biológicas e nas lipoproteínas. As alterações estruturais dos fosfolípidos, ocorrem devido ao stress oxidativo e podem manisfestar-se em alterações das suas propriedades e funções. Já são conhecidas algumas condições fisiopatológicas, nas quais os fosfolípidos oxidados estão envolvidos, por exemplo sinalização celular, resposta imunitária, apoptose e doenças relacionadas com o envelhecimento. Por esse motivo, o interesse no estudo dos fosfolípidos oxidados e suas funções tem crescido nos últimos anos. Contudo, a maioria dos estudos realizados, focam a oxidação das fosfatidilcolinas, tendo sido dedicada pouca atenção a outras classes de fosfolipídos, como as fosfatidiletanolaminas. As fosfatidiletanolaminas, podem ainda sofrer outras modificações, devido ao grupo amina livre presente na cabeça polar, como por exemplo a glicação. As fosfatidiletanolaminas glicadas já foram detectadas em condições de hiperglicemia, em pacientes diabéticos, e tem correlação com a hemoglobina glicada. Sabe-se que a glicação de biomoléculas, pode aumentar as modificações oxidativas, que por sua vez, podem ser responsáveis pelo estado inflamatório, existente na diabetes mellitus. Tanto o stress oxidativo, como a inflamação estão relacionados com a diabetes e as suas complicações. A espectrometria de massa tem sido utilizada como uma importante tecnologia na detecção e caracterização de modificações oxidativas de fosfolípidos. Assim, neste trabalho pretendeu-se estudar as modificações oxidativas induzidas em fosfatidiletanolaminas glicadas, e os seus efeitos biológicos nos monócitos e células dendríticas do sangue periférico. Pretendeu-se ainda, estudar as alterações que ocorreram nas espécies de fosfatidiletanolaminas do fígado de ratos diabéticos. Os resultados obtidos permitiram identificar vários produtos de oxidação de fosfatidiletanolaminas glicadas, nomeadamente novos produtos formados pela oxidação da cabeça polar glicada. A oxidação na cabeça polar glicada foi, ainda, confirmada pela realização de experiências com spin traps combinadas com espetrometria de massa. Posteriormente, as fosfatidiletanolaminas oxidadas, glicadas e glicoxidadas demonstraram ter efeitos pró-inflamatórios, confirmados pelo aumento da estimulação monócitos e de células dendríticas, expresso no aumento do número de células produtoras de citocinas em comparação com o estado basal. As diferentes modificações de fosfatidiletanolaminas induziram estímulos distintos nos dois tipos de células. Sendo as fosfatidiletanolaminas glicadas e as glicoxidadas, os compostos que induziram um maior estímulo. Estes resultados sugeriram que as fosfatidiletanolaminas glicadas e as glicoxidadas podem estar associadas com o estado inflamatório que decorre da hiperglicemia crónica. Ainda, a avaliação do perfil lipídico de extratos de fígado de ratos diabéticos demonstrou que a hiperglicemia induz inúmeras alterações das espécies de fosfatidiletanolaminas e das espécies de outras classes de fosfolípidos, em simultâneo com sinais de lesão hepática. Em conclusão, este trabalho demonstra a relação existente entre, a hiperglicémia, o stress oxidativo, a glicação e oxidação de fosfatidiletanolaminas e ainda a inflamação e compliações diabéticas. Portanto a contribuição da lipidómica é importante para compreender os efeitos prejudiciais da hiperglicemia não controlada, e por isso, merece ser mais explorado.

Metabolic signature of lung cancer: a metabolomic study of human tissues and biofluids

This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.

Evaluation of the matabolic response of osteosarcoma cells to conventional and new anticancer drugs by NMR metabolomics

The main scope of this work was to evaluate the metabolic effects of anticancer agents (three conventional and one new) in osteosarcoma (OS) cells and osteoblasts, by measuring alterations in the metabolic profile of cells by nuclear magnetic resonance (NMR) spectroscopy metabolomics. Chapter 1 gives a theoretical framework of this work, beginning with the main metabolic characteristics that globally describe cancer as well as the families and mechanisms of action of drugs used in chemotherapy. The drugs used nowadays to treat OS are also presented, together with the Palladium(II) complex with spermine, Pd2Spm, potentially active against cancer. Then, the global strategy for cell metabolomics is explained and the state of the art of metabolomic studies that analyze the effect of anticancer agents in cells is presented. In Chapter 2, the fundamentals of the analytical techniques used in this work, namely for biological assays, NMR spectroscopy and multivariate and statistical analysis of the results are described. A detailed description of the experimental procedures adopted throughout this work is given in Chapter 3. The biological and analytical reproducibility of the metabolic profile of MG-63 cells by high resolution magic angle spinning (HRMAS) NMR is evaluated in Chapter 4. The metabolic impact of several factors (cellular integrity, spinning rate, temperature, time and acquisition parameters) on the 1H HRMAS NMR spectral profile and quality is analysed, enabling the definition of the best acquisition parameters for further experiments. The metabolic consequences of increasing number of passages in MG-63 cells as well as the duration of storage are also investigated. Chapter 5 describes the metabolic impact of drugs conventionally used in OS chemotherapy, through NMR metabolomics studies of lysed cells and aqueous extracts analysis. The results show that MG-63 cells treated with cisplatin (cDDP) undergo a strong up-regulation of lipid contents, alterations in phospholipid constituents (choline compounds) and biomarkers of DNA degradation, all associated with cell death by apoptosis. Cells exposed to doxorubicin (DOX) or methotrexate (MTX) showed much slighter metabolic changes, without any relevant alteration in lipid contents. However, metabolic changes associated with altered Krebs cycle, oxidative stress and nucleotides metabolism were detected and were tentatively interpreted at the light of the known mechanisms of action of these drugs. The metabolic impact of the exposure of MG-63 cells and osteoblasts to cDDP and the Pd2Spm complex is described in Chapter 6. Results show that, despite the ability of the two agents to bind DNA, the metabolic consequences that arise from exposure to them are distinct, namely in what concerns to variation in lipid contents (absent for Pd2Spm). Apoptosis detection assays showed that, differently from what was seen for MG-63 cells treated with cDDP, the decreased number of living cells upon exposure to Pd2Spm was not due to cell death by apoptosis or necrosis. Moreover, the latter agent induces more marked alterations in osteoblasts than in cancer cells, while the opposite seemed to occur upon cDDP exposure. Nevertheless, the results from MG-63 cells exposure to combination regimens with cDDP- or Pd2Spm-based cocktails, described in Chapter 7, revealed that, in combination, the two agents induce similar metabolic responses, arising from synergy mechanisms between the tested drugs. Finally, the main conclusions of this thesis are summarized in Chapter 8, and future perspectives in the light of this work are presented.

Lipidomic analysis of mesenchymal cells candidates for cell therapy

Mesenchymal stromal cells are adult stem cells found mostly in the bone marrow. They have immunosuppressive properties and they have been successfully applied as biological therapy in several clinical trials regarding autoimmune diseases. Despite the great number of clinical trials, MSCs’ action is not fully understand and there are no identified markers that correlate themselves with the immunomodulatory power. A lipidomic approach can solve some of these problems once lipids are one of the major cells’ components. Therefore, in this study cells’ lipidome was analysed and its deviations were evaluated according to the medium of culture and to the presence of pro-inflammatory stimuli, mimicking physiological conditions in which these cells are used. This was the first study ever made that aimed to analyse the differences in the phospholipid profile between mesenchymal stromal cells non-stimulated and stimulated with proinflammatory stimulus. This analysis was conducted in both cells cultured in medium supplemented with animal serum and in cells cultured in a synthetic medium. In cells cultured in the standard medium the levels of phosphatidylcholine (PC) species with shorter fatty acids (FAs) acyl chains decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of lysoPC (LPC)(18:0) - an anti-inflammatory LPC - observed in cells subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN- γ also enhanced the levels of phosphatidylethanolamine PE(40:6) and decreased the levels of PE(38:6). Higher expression of phosphatidylserine PS(36:1) and sphingomyelin SM(34:0) along with a decrease in PS(38:6) levels were observed. However, in cells cultured in a synthetic medium, TNF-α and IFN-γ only enhanced the levels of PS(36:1). These results indicate that lipid metabolism and signaling is modulated during mesenchymal stromal cells action.

Estudo da aplicabilidade de biossensores piezoelécticos para a detecção de lipopolissacáridos bacterianos

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

On the solvation in lipid bilayers measured by pyrene fluorescence: thermal and molecular composition influence on equivalent polarity in lipidic mixtures

Phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol (CHOL) are major constituents of mammalian cell membranes. DPPC/CHOL and DPPC/DMPC are well-known binary mixtures. POPC/CHOL, DOPC/CHOL, egg-SM/CHOL, egg-SM/POPC and egg-SM/DOPC are less studied, but also important for the comprehension of the POPC/egg-SM/CHOL mixtures. These provide complex media for which polarity is hard to access. It is mainly determined by the water penetrating the bilayer (unevenly distributed creating a polarity gradient), though the influence of the dipoles from phospholipids (e.g. –PO, –CO, –OH) and the double bond in the steroid ring of CHOL cannot be neglected. CHOL derivatives are an interesting tool to verify the influence of the double bonds in the polarization of its surroundings. Pyrene fluorescence was used to access an equivalent polarity (associated to the dielectric constant) near the lipid/water interface of lipid bilayers. POPC/CHOL and DOPC/CHOL have similar thermal behavior and variation with CHOL content, though for lower CHOL content the equivalent polarity is higher for the DOPC/CHOL mixtures. The studies with DPPC and DMPC showed that pyrene does not seem to have a marked preference for either ordered or disordered phases. For DPPC/CHOL and egg-SM/CHOL the highlight goes to the behavior of the mixtures at higher CHOL amounts, where there is a substantial change in the thermal behavior and polarity values especially for the egg-SM/CHOL mixture. Egg-SM/POPC and egg-SM/DOPC show different behavior depending on which phospholipid has a higher molar proportion. The ternary mixtures analyzed do not exhibit significant differences, though there is the indication of the existence of a more ordered environment at lower temperatures and a less ordered environment for higher temperatures. The presence of 7DHC or DCHOL in egg-SM bilayers showed a tendency for the same behavior detected upon mixing higher amounts of CHOL.

Contribution to fundamental aspects of biophysics, radiobiology, and modeling of cellular response to low radiation doses

Tese de doutoramento, Engenharia Biomédica e Biofísica, Universidade de Lisboa, Faculdade de Ciências, 2014

Modulation of calcitonin amyloid formation by anionic lipid membranes

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014

Synaptic dysfunction in early encephalopathies : from genes to function

Tese de doutoramento, Medicina (Neurologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Increasing the wear resistance of molds for injection of glass fiber reinforced plastics

Abrasion by glass fibers during injection molding of fiber reinforced plastics raises new challenges to the wear performance of the molds. In the last few decades, a large number of PVD and CVD coatings have been developed with the aim of minimizing abrasion problems. In this work, two different coatings were tested in order to increase the wear resistance of the surface of a mold used for glass fiber reinforced plastics: TiAlSiN and CrN/CrCN/DLC. TiAlSiN was deposited as a graded monolayer coating while CrN/CrCN/DLC was a nanostructured coating consisting of three distinct layers. Both coatings were produced by PVD unbalanced magnetron sputtering and were characterized using scanning electron microscopy (SEM) provided with energy dispersive spectroscopy (EDS), atomic force microscopy (AFM), micro hardness (MH) and scratch test analysis. Coating morphology, thickness, roughness, chemical composition and structure, hardness and adhesion to the substrate were investigated. Wear resistance was characterized through industrial tests with coated samples and an uncoated reference sample inserted in a feed channel of a plastic injection mold working with 30 wt.% glass fiber reinforced polypropylene. Results after 45,000 injection cycles indicate that the wear resistance of the mold was increased by a factor of 25 and 58, by the TiAlSiN and CrN/CrCN/DLC coatings, respectively, over the uncoated mold steel.

Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-Screen Printed Electrodes

A gold screen printed electrode (Au-SPE) was modified by merging Molecular Imprinting and Self-Assembly Monolayer techniques for fast screening cardiac biomarkers in point-of-care (POC). For this purpose, Myoglobin (Myo) was selected as target analyte and its plastic antibody imprinted over a glutaraldehyde (Glu)/cysteamine (Cys) layer on the gold-surface. The imprinting effect was produced by growing a reticulated polymer of acrylamide (AAM) and N,N′-methylenebisacrylamide (NNMBA) around the Myo template, covalently attached to the biosensing surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies were carried out in all chemical modification steps to confirm the surface changes in the Au-SPE. The analytical features of the resulting biosensor were studied by different electrochemical techniques, including EIS, square wave voltammetry (SWV) and potentiometry. The limits of detection ranged from 0.13 to 8 μg/mL. Only potentiometry assays showed limits of detection including the cut-off Myo levels. Quantitative information was also produced for Myo concentrations ≥0.2 μg/mL. The linear response of the biosensing device showed an anionic slope of ~70 mV per decade molar concentration up to 0.3 μg/mL. The interference of coexisting species was tested and good selectivity was observed. The biosensor was successfully applied to biological fluids.

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

3D-nanostructured Au electrodes for the event-specific detection of MON810 transgenic maize

In the present work, the development of a genosensor for the event-specific detection of MON810 transgenic maize is proposed. Taking advantage of nanostructuration, a cost-effective three dimensional electrode was fabricated and a ternary monolayer containing a dithiol, a monothiol and the thiolated capture probe was optimized to minimize the unspecific signals. A sandwich format assay was selected as a way of precluding inefficient hybridization associated with stable secondary target structures. A comparison between the analytical performance of the Au nanostructured electrodes and commercially available screen-printed electrodes highlighted the superior performance of the nanostructured ones. Finally, the genosensor was effectively applied to detect the transgenic sequence in real samples, showing its potential for future quantitative analysis.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.