Applications of Graphene Quantum Dots in Biomedical Sensors

Due to the proliferative cancer rates, cardiovascular diseases, neurodegenerative disorders, autoimmune diseases and a plethora of infections across the globe, it is essential to introduce strategies that can rapidly and specifically detect the ultralow concentrations of relevant biomarkers, pathogens, toxins and pharmaceuticals in biological matrices. Considering these pathophysiologies, various research works have become necessary to fabricate biosensors for their early diagnosis and treatment, using nanomaterials like quantum dots (QDs). These nanomaterials effectively ameliorate the sensor performance with respect to their reproducibility, selectivity as well as sensitivity. In particular, graphene quantum dots (GQDs), which are ideally graphene fragments of nanometer size, constitute discrete features such as acting as attractive fluorophores and excellent electro-catalysts owing to their photo-stability, water-solubility, biocompatibility, non-toxicity and lucrativeness that make them favorable candidates for a wide range of novel biomedical applications. Herein, we reviewed about 300 biomedical studies reported over the last five years which entail the state of art as well as some pioneering ideas with respect to the prominent role of GQDs, especially in the development of optical, electrochemical and photoelectrochemical biosensors. Additionally, we outline the ideal properties of GQDs, their eclectic methods of synthesis, and the general principle behind several biosensing techniques.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz

Aptamer-modified nanomaterials: Principles and applications

Aptamers are promising alternative binders that can substitute antibodies in various applications. Due to the advantages of aptamers, namely their high affinity, specificity and stability, along with the benefits originating from the chemical synthesis of aptamers, they have attracted attention in various applications including their use on nanostructured material. This necessitates the immobilization of aptamers on a solid support. Since aptamer immobilization may interfere with its binding properties, the immobilization of aptamers has to be investigated and optimized. Within this review, we give general insights into the principles and factors controlling the binding affinity of immobilized aptamers. Specific features of aptamer immobilization on nanostructured surfaces and nanoparticles are highlighted and a brief overview of applications of aptamer-modified nanostructured materials is given

Nanoprobes for Tumor Theranostics

This book reports cutting-edge technology in nanoprobes or nanobiomaterials used for the accurate diagnosis and therapy of tumors, involving a multidisciplinary of chemistry, materials science, oncology, biology, and medicine

New nanotechnology approaches using dendrimers modified with natural polymers for controlling stem cells behaviour in tissue engineering strategies

Tese de doutoramento em Ciência e Tecnologia de Materiais (ramo de conhecimento em Engenharia de Tecidos - Materiais Híbridos)In the recent years, great progress has been done in the emerging field of tissue engineering. Despite the important advances the performance of cells-scaffold constructs, one of the several tissue engineering approaches, remains limited in part due to the need for optimize cell culture techniques and culture media. Nanocarrier systems have generated a significant amount of interest in the ex vivo cell maintenance, and control of the cellular fate in vivo mainly due to their internalization efficiency, drug loading capacity, and to favorably modulate the solubility and pharmacokinetics of drugs. Dendrimers are synthetic, monodispersive, spherical and highly branched macromolecules that present unique advantages and fulfills most requirements as carriers for drug delivery; however, it has been found that high generation dendrimers are often cytotoxic. Thus, in this thesis we focused our attention in this fundamental problem and explore the development of novel nanobiomaterials based on the grafting of carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers, the socalled CMCht/PAMAM dendrimer nanoparticles. These macromolecular vehicles were developed to explore a new concept consisting on the intracellular and controlled delivery of bioactive molecules aimed at control stem cells functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. Thus, by combining nanotechnology-based systems and traditional tissue engineering strategies, we expect to develop a novel therapeutic solution for the efficient treatment of damage/diseased cells and tissues. To validate this new concept, there is the need to evaluate the performance of the developed nanocarriers, in vitro and in vivo. Firstly, the uptake efficiency and internalization mechanism of fluorescent-labeled CMCht/PAMAM dendrimer nanoparticles was investigated using different cell types. Fluorescence microscopy studies revealed that the developed nanocarriers could be efficiently internalized, either by cell lines and primary cultures, after a few hours. Flow cytometry studies revealed that rat bone marrow stromal cells (RBMSCs) cultured in the presence of colchicine, an alkaloid that inhibits endocytosis, decreased the internalization of the nanoparticles. These data showed that uptake by cells was primarily via an active endocytotic mechanism, but not exclusively. Preliminary studies were also carried out to evaluate the possible applicability of the CMCht/PAMAM dendrimer nanoparticles in the central nervous system. Internalization rate, cell viability and metabolic activity studies were performed using rat post-natal hippocampal neurons and cortical glial cells that revealed their ability for being taken up by these cells and its non cytotoxicity. Complementarily, dexamethasone (Dex), a glucocorticoid known to have important role on the proliferation and expression of osteoblastic differentiation markers, was used as a model drug and incorporated into the bulk of the nanoparticles. Physicochemical characterization and in vitro biological studies have demonstrated that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles were successfully synthesized, were not cytotoxic in the range of concentration below 1 mg.mL-1 and promote osteogenesis (2-D system). To assess the true value of the Dex-loaded CMCht/PAMAM dendrimer nanoparticles systems for application in tissue engineering strategies, we use different biomaterials to develop a set of novel scaffolds both ceramic and polymeric or formulations. These scaffolds were found to be suitable for applications in bone, cartilage and osteochondral tissue engineering. In vitro studies have shown that combination of scaffolds, bone marrow stromal cells and Dex-loaded CMCht/PAMAM dendrimer nanoparticles (3-D system) enhanced osteogenesis. Finally, in vivo studies have shown that the novel Dex-loaded CMCht/PAMAM dendrimer nanoparticles may be beneficial as intracellular nanocarrier, supplying Dex in a regimented manner, while avoiding the need of culturing stem cells for long periods of time in vitro, towards promoting the osteogenic differentiation. Remarkably, the proposed strategy allow modulate and direct stem cells differentiation towards osteogenic phenotype, enhance in vivo proteoglycan extracellular matrix synthesis and promote superior de novo bone formation. This thesis mark the transition from the ‘proof-of-concept’ to useful intracellular nanocarrier tool, as the Dex-loaded CMCht/PAMAM dendrimer nanoparticles show promise for application in direct stem cell to a particular cell fate, in vitro and in vivo.Grandes progressos têm sido feitos nos últimos tempos no emergente campo científico da engenharia de tecidos. No entanto, a eficiência dos sistemas células-matriz tridimensional porosa, uma das estratégias usadas nas abordagens em engenharia de tecidos, tem sido limitada, em parte, pela necessidade de se optimizarem as técnicas e os meios de cultura usados. Sistemas de nanopartículas para o transporte de moléculas bioactivas têm suscitado grande interesse na área da biomedicina, nomeadamente no que respeita à possível aplicação como suplementos em meios de cultura ex vivo, e no controlo das funções celulares in vivo. Isto deve-se, principalmente, à sua eficiência de internalização, elevada capacidade de incorporação de fármacos, e ao facto de favorecerem a solubilidade de moléculas hidrofóbicas e de possibilitarem a modulação da sua farmacocinética. Os dendrímeros são sistemas macromoleculares sintéticos altamente ramificados que apresentam características únicas, tais como monodispersividade e uma estrutura esférica, e que preenchem a maioria dos requisitos para serem usados como veículos para a libertação controlada de fármacos. Não obstante, tem-se verificado que dendrímeros de elevada geração apresentam tipicamente uma citotoxicidade indesejada. Assim, nesta tese a atenção focou-se na resolução deste problema fundamental. Para tal foi explorado o desenvolvimento de novos nanobiomateriais tendo como estratégia a ligação química do polímero carboximetilquitosano (CMCht) a dendrímeros de poliamidoamina (PAMAM) de baixa geração, que se denominaram por nanopartículas de carboximetilquitosano/poliamidoamina (CMCht/PAMAM). Estas macromoléculas foram desenvolvidas e um novo conceito aplicativo foi testado relacionado com a sua aplicação em medicina regenerativa como veículos de libertação controlada e intracelular de moléculas bioactivas, de forma a ser possível controlar efectivamente as actividades celulares, tais como a proliferação e a diferenciação de células estaminais ex vivo, e a manutenção do fenótipo dessas mesmas células após o seu implante. Assim, recorrendo à nanotecnologia e a estratégias usadas na engenharia de tecidos, espera-se que seja possível desenvolver uma nova solução terapêutica que possa possibilitar, de uma forma eficiente, o tratamento de células e tecidos danificados ou que apresentem algum tipo de patologia. De forma a validar este novo conceito, é necessário avaliar o potencial dos nanosistemas desenvolvidos, in vitro e in vivo. Primeiramente, a eficiência e o mecanismo de internalização de nanopartículas de CMCht/PAMAM ligadas a um marcador fluorescente foram investigados, recorrendo a estudos celulares, in vitro. Estudos de microscopia de fluorescência revelaram que as nanopartículas desenvolvidas são internalizadas por diferentes tipos de células após algumas horas em cultura, incluindo linhas celulares e culturas primárias. Estudos envolvendo a citometria de fluxo mostraram que quando células multipotentes do estroma da medula óssea de rato (RBMSCs), foram cultivadas na presença de colchicina, um alcalóide inibidor da endocitose, exibiam menor capacidade de internalização das nanopartículas. Assim, a internalização das nanopartículas pelas células ocorre principalmente por um mecanismo de endocitose, mas que este não é o único. Foram também realizados estudos para determinar a taxa de internalização, viabilidade celular e actividade metabólica, recorrendo a neurónios isolados do hipocampo de rato pós-natais e células da glia. Estes estudos mostraram que as nanopartículas são internalizadas por estas células, e não afectam negativamente a viabilidade da cultura das células. A dexametasona (Dex), uma molécula pertencente à família dos glucocorticóides e conhecida pelo seu papel na modulação da proliferação e expressão dos marcadores de diferenciação osteoblástica, foi usada como fármaco modelo e incorporada nas nanopartículas de CMCht/PAMAM. A caracterização físico-química e estudos biológicos in vitro demonstraram que as nanopartículas de CMCht/PAMAM carregadas com Dex foram sintetizadas com sucesso, não apresentaram citotoxicidade em concentrações até 1 mg.mL-1 e promoveram a osteogénese (sistema de cultura 2-D). Por forma a avaliar o verdadeiro potencial aplicativo das nanopartículas de CMCht/PAMAM carregadas com Dex em estratégias de engenharia de tecidos, diferentes biomateriais foram usados no desenvolvimento de estruturas tridimensionais porosas, incluindo cerâmicos, polímeros e formulações contendo ambos. Estas estruturas tridimensionais porosas mostraram-se adequadas para serem usadas em engenharia de tecidos de osso, cartilagem e defeitos osteocondrais. Estudos in vitro revelaram que a abordagem constituída por estruturas tridimensionais porosas, RBMSCs e nanopartículas de CMCht/PAMAM carregadas com Dex (sistema de cultura 3-D) promoveu um aumento significativo da osteogénese. Por último, estudos in vivo mostraram que as nanopartículas de CMCht/PAMAM carregadas com Dex são um sistema de libertação intracelular altamente eficiente, uma vez que possibilitaram a libertação de Dex com um perfil cinético adequado, permitindo assim evitar os longos períodos de cultura in vitro, necessários à diferenciação osteogénica de células estaminais. A estratégia proposta permite modular e direccionar a diferenciação das células estaminais para o fenótipo osteogénico, aumentar a síntese de proteoglicanos da matriz extracelular e promover a formação de osso num estado de maturação mais avançado. Esta tese marca a transição de “prova de conceito” para uma ferramenta aplicativa das nanopartículas desenvolvidas na libertação intracelular de fármacos, uma vez que as nanopartículas de CMCht/PAMAM carregadas com Dex se mostraram promissoras no direccionamento das células estaminais para um determinado fenótipo, in vitro e in vivo.Fundação para a Ciência e a Tecnologia (FCT) - ref. SFRH/BD/21786/2005 através dos programas POCTI e FEDERRotary Club de Caldas das TaipasFundação para a Ciência e Tecnologia (FCT) - PhD grant Ref. SFRH/BD/21786/2005, POCTI, FEDER programsCanon Foundation in EuropeEuropean NoE EXPERTISSUES (NMP3-CT-2004-500283)European Union HIPPOCRATES STREP Project (NMP3-CT-2003-505758

Aptamer-based optical biosensors

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Literature-based discovery of known and potential new mechanisms for relating the status of cholesterol to the progression of breast cancer

Breast cancer has been studied for a long period of time and from a variety of perspectives in order to understand its pathogeny. The pathogeny of breast cancer can be classified into two groups: hereditary and spontaneous. Although cancer in general is considered a genetic disease, spontaneous factors are responsible for most of the pathogeny of breast cancer. In other words, breast cancer is more likely to be caused and deteriorated by the dysfunction of a physical molecule than be caused by germline mutation directly. Interestingly, cholesterol, as one of those molecules, has been discovered to correlate with breast cancer risk. However, the mechanisms of how cholesterol helps breast cancer progression are not thoroughly understood. As a result, this study aims to study known and discover potential new mechanisms regarding to the correlation of cholesterol and breast cancer progression using literature review and literature-based discovery. The known mechanisms are further classified into four groups: cholesterol membrane content, transport of cholesterol, cholesterol metabolites, and other. The potential mechanisms, which are intended to provide potential new treatments, have been identified and checked for feasibility by an expert

Drug Delivery to Solid Tumors via Polymeric Nanoparticles

A main challenge in chemotherapy is to deliver an anti-cancer drug selectively to tumor and avoid off-target exposure to other body tissues and organs. Nanoparticles (NPs) have been considered a promising approach for tumor drug delivery, with popularity attributable to the famous “Enhanced Permeability and Retention effect”, where small particles enter tumor tissues through leaky vasculature and be retained there. Currently, the phagocytic clearance of NPs is avoided by coating NP surface with Polyethylene glycol (PEG). Although successful in prolonging NPs circulation, PEG prevents proper interaction of NPs with the target cells, known as “PEG dilemma”. Low molecular weight chitosan (LMWC) can function as a hydrophilic pH-sensitive alternative stealth coating for NPs. The LMWC-coated NPs were previously made with a conjugate of poly(lactide-co-glycolic) acid (PLGA) and LMWC (PLGA-LMWC) and showed pH-sensitive surface charge. However, this preparation method has disadvantages such as production complexity and difficulty in drug encapsulation. We used an alternative surface modification method based on dopamine polymerization, which formed a layer of polydopamine (pD) on NP surface allowing for conjugation of LMWC to the preformed NP cores. When compared to PLGA-LMWC NPs, PLGA-pD-LMWC NPs had superior control over drug release. Additionally, obtained PLGA-pD-LMWC NPs had similar cellular interactions to that of PLGA-LMWC NPs, achieving cellular uptake in cancer cells under mildly acidic conditions, which was not achieved with PEG coated NPs. However, when tested in vivo, there was no significant difference between LMWC and PEG-coated NPs in terms of tumor growth suppression and tumor accumulation. While the exact reason behind the poor in vitro-in vivo correlation remain be confirmed, it is hypothesized to be premature drug release or protein corona formation

Non-covalent interactions in organotin(IV) derivatives of 5,7-ditertbutyl- and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine as recognition motifs in crystalline self- assembly and their in vitro antistaphylococcal activity

Non-covalent interactions are known to play a key role in biological compounds due to their stabilization of the tertiary and quaternary structure of proteins [1]. Ligands similar to purine rings, such as triazolo pyrimidine ones, are very versatile in their interactions with metals and can act as model systems for natural bio-inorganic compounds [2]. A considerable series (twelve novel compounds are reported) of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl- 1,2,4-triazolo[1,5-a]pyrimidine (dptp) were synthesized and investigated by FT-IR and 119Sn M\uf6ssbauer in the solid state and by 1H and 13C NMR spectroscopy, in solution [3]. The X-ray crystal and molecular structures of Et2SnCl2(dbtp)2 and Ph2SnCl2(EtOH)2(dptp)2 were described, in this latter pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the -OH group of the ethanol moieties. The network of hydrogen bonding and aromatic interactions involving pyrimidine and phenyl rings in both complexes drives their self-assembly. Noncovalent interactions involving aromatic rings are key processes in both chemical and biological recognition, contributing to overall complex stability and forming recognition motifs. It is noteworthy that in Ph2SnCl2(EtOH)2(dptp)2 \u3c0\u2013\u3c0 stacking interactions between pairs of antiparallel triazolopyrimidine rings mimick basepair interactions physiologically occurring in DNA (Fig.1). M\uf6ssbauer spectra suggest for Et2SnCl2(dbtp)2 a distorted octahedral structure, with C-Sn-C bond angles lower than 180\ub0. The estimated angle for Et2SnCl2(dbtp)2 is virtually identical to that determined by X-ray diffraction. Ph2SnCl2(EtOH)2(dptp)2 is characterized by an essentially linear C-Sn-C fragment according to the X-ray all-trans structure. The compounds were screened for their in vitro antibacterial activity on a group of reference staphylococcal strains susceptible or resistant to methicillin and against two reference Gramnegative pathogens [4] . We tested the biological activity of all the specimen against a group of staphylococcal reference strains (S. aureus ATCC 25923, S. aureus ATCC 29213, methicillin resistant S. aureus 43866 and S. epidermidis RP62A) along with Gram-negative pathogens (P. aeruginosa ATCC9027 and E. coli ATCC25922). Ph2SnCl2(EtOH)2(dptp)2 showed good antibacterial activity with a MIC value of 5 \u3bcg mL-1 against S. aureus ATCC29213 and also resulted active against methicillin resistant S. epidermidis RP62A