Exploiting double exchange Diels-Alder cycloadditions for immobilization of peptide nucleic acids on gold nanoparticles

The generation of PNA-decorated gold nanoparticles (AuNPs) has revealed to be more difficult as compared to the generation of DNA-functionalized ones. The less polar nature of this artificial nucleic acid system and the associated tendency of the neutral poly-amidic backbone to aspecifically adsorb onto the gold surface rather than forming a covalent bond through gold-thiol interaction, combined with the low solubility of PNAs itself, form the main limiting factors in the functionalization of AuNP. Here, we provide a convenient methodology that allows to easily conjugate PNAs to AuNP. Positively charged PNAs containing a masked furan moiety were immobilized via a double exchange Diels-Alder cycloaddition onto masked maleimide-functionalized AuNPs in a one-pot fashion. Conjugated PNA strands retain their ability to selectively hybridize with target DNA strands. Moreover, the duplexes resulting from hybridization can be detached through a retro-Diels-Alder reaction, thus allowing straightforward catch-and-release of specific nucleic acid targets

Biophysics of DNA based Nanosystems Probed by Optical Nanoscopy

A dynamic DNA nanosystem exploits the programmable structure and energy landscape of DNA self-assembly to encode designed processes in a fuctuating molecular environment. One type of such a dynamic system, DNA walker, is reminiscent of biological motor proteins that convert chemical energy into mechanical translocation. Typical DNA walker travels tens of nanometers at a speed orders of magnitude slower than motor proteins. Two major challenges limited the development of functional DNA walkers. First, there are no suitable characterization methods that o˙er adequate spatial and temporal resolution to extract walker kinetics. Second, no guidelines have been established for the design and development of DNA walkers with specifed properties. In this work, an enzymatic DNA walker system that integrate oligonucleotides with nanomaterials is designed. This approach takes advantage of novel optical properties of nanomaterials and sub-di˙raction imaging techniques to study the kinetics and biophysical nature of synthetic DNA walkers. Design principles are extracted from walker kinetics for constructing functional walkers that can rival motor proteins. Multiple schemes are explored to regulate the walker motility so that various behaviors can be encoded into the system. This work demonstrates novel methods to design and construct molecular systems with programmed functions, which will pave the road for creating synthetic systems with encoded behaviors from the bottom up

Competitive Activation of a Methyl C−H Bond of Dimethylformamide at an Iridium Center

During the synthesis of [AsPh_4][Ir(CO)_2I_3Me] by refluxing IrCl_3·3H_2O in DMF (DMF = dimethylformamide) in the presence of aqueous HCl, followed by sequential treatment with [AsPh_4]Cl, NaI, and methyl iodide and finally recrystallization from methylene chloride/pentane, three crystalline byproducts were obtained: [AsPh4]_2[Ir(CO)I_5], [AsPh_4]_2[trans-Ir(CO)I_4Cl], and [AsPh_4][Ir(CO)(κ^2O,C-CH_2NMeCHO)Cl_2I]. The last of these, whose structure (along with the others) was determined by X-ray diffraction, results from activation of a methyl C−H bond of dimethylformamide, rather than the normally much more reactive aldehydic C−H bond

Modular Nucleic Acid Assembled p/MHC Microarrays for Multiplexed Sorting of Antigen-Specific T Cells

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called “Nucleic Acid Cell Sorting (NACS)”, single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection

Optimization of chitosan-based nanosystems to deliver plasmid DNA vaccines

Cancer is the second leading cause of death worldwide and it is estimated that soon it could become the first. Cervical cancer is the fourth most common cancer among women worldwide and in underdeveloped countries, it is the most common cancer among women. This cancer is most often caused by the Human Papilloma Virus (HPV). The incidence of this cancer has decreased in countries with good health systems due to a vaccination and prevention programme against this virus. Currently, vaccines are based on viruslike particles constructed from the recombinant expression of the L1 protein, which belongs to the HPV capsid. However, existing vaccines are only preventive and have no th erapeutic effect if the vaccine is administered to a person who has previously contracted the infection. Currently , DNA vaccines constitute an innovative tool with great potential to help in the battle against viral infections or from other pathogens , and against various types of cancer. To deliver the DNA to eukaryotic cells, a delivery system must be created to protect, transport and deliver the DNA to the target cells. DNA vaccines aim to encode genes, characteristic of pathogens or cancer cells, in orde r to activate and generate specific immune responses. In this context, the development of a DNA vaccine encoding the HPV E7 oncoprotein, responsible for interfering with the tumour suppressor retinoblastoma protein, which is responsible for regulating the cell cycle of eukaryotic cells, may be an effective tool against HPV potential and therapeuticinduced cervical cancer, due to its preventive effects. To maximise the efficiency of these vaccines, the development of an appropriate delivery system is critic al. Thus, the present work was based on the optimization of the formulation of delivery systems based on the chitosan polymer, and four types of chitosan with different molecular weights were explored: high molecular weight chitosan (HMW, 200500 kDa), low molecular weight (LMW, 50 to formulate the systems was the 190 kDa), 20 kDa and 5 kDa. The technique used ionotropic gelation, based on ionic crosslinking. The nanoparticle formulation resulted from the interaction positively charg ed chitosan polymers, and the established negatively charged between the crosslinker sodium tripolyphosphate (TPP) and the plasmid DNA (pADN), encoding mutated E7. The inclusion of TPP aims to compact the formed nanoparticles and increase their stability , thus favouring a more de fined round shape. To achieve the best delivery systems, a design of experiments (DoE) tool was used. This tool allowed combining several parameters/inputs simultaneously (chitosan and TPP concentrations) to obtain the optimal formulation conditions of the systems based on the chosen responses (size, polydispersity index (PDI) and zeta potential). After performing the proposed tests and characterizing the properties of the obtained formulations, the respective responses were introduced in the DoE program an d a statistical analysis was performed. The linear and quadratic models obtained were statistically significant (pvalue <0,05) and the "lack of al points for each chi fit" was not significant, with an adequate determination coefficient. The predicted optim tosan polymer were all successfully validated. Subsequently, further studies were conducted to evaluate the properties of the delivery systems formulated with the conditions defined at the optim al points. Scanning electron microscopy (SEM) analyses were performed to evaluate the morphology, Fourier transform infrared spectroscopy was used to evaluate the chemical properties and specific functional groups, while different stability tests were perfor med to evaluate the strength and DNA release, and finally cytotoxicity tests were performed to ensure the biocompatibility of the chitosan nanoparticles. The four delivery systems developed , presented satisfactory characteristics , such as spherical or oval stability and strength, DNA retention and good biocompatibility. nanoparticles, good In this sense, the DoE tool proved to be a powerful tool to explore and tailor the characteristics of chitosan/TPP/pADN nanosystems, allowing the development of a system suitable for the encapsulation, transport and delivery of pADN, thus providing the advancement of the DNA vaccine delivery research field.O cancro é a segunda causa de morte a nível mundial e estima-se que, num futuro próximo, poderá tornar-se a primeira. O cancro do colo do útero é o quarto cancro mais comum entre as mulheres em todo o mundo, e em países pouco desenvolvidos, chega a ser o que tem a maior incidência entre as mulheres. Este tipo de cancro é causado, na maioria das vezes, pelo Vírus do Papiloma Humano (HPV). A incidência deste cancro diminuiu em países com bons sistemas de saúde devido a um programa de vacinação e prevenção contra este vírus. As vacinas administradas atualmente são baseadas em partículas semelhantes a vírus, construídas a partir da expressão recombinante da proteína L1, que pertence ao capsídeo do HPV. No entanto, estas vacinas são apenas preventivas, não tendo qualquer efeito terapêutico caso a vacina seja administrada a uma pessoa que tenha contraído previamente a infeção. Atualmente, as vacinas de ADN têm sido uma ferramenta inovadora com grande potencial para ajudar na batalha contra infeções virais, ou provenientes de outros agentes patogénicos, e contra vários tipos de cancro. Para entregar o ADN às células eucarióticas, deve ser criado um sistema de entrega para proteger, transportar e entregar o ADN às célula-alvo. As vacinas de ADN têm como objetivo codificar genes, característicos dos agentes patogénicos ou de células cancerígenas, de forma a ativar e gerar respostas imunitárias específicas. Neste contexto, o desenvolvimento de uma vacina de ADN que codifica a oncoproteína E7 do HPV pode ser uma ferramenta eficaz contra o cancro do colo do útero, induzido pela infeção do vírus HPV, devido aos seus potenciais efeitos preventivos e terapêuticos. Esta oncoproteína é responsável por interferir com a proteína do retinoblastoma, que por sua vez, desempenha funções na regulação do ciclo celular das células eucarióticas. Para maximizar a eficiência destas vacinas, é fundamental o desenvolvimento de um sistema de entrega adequado. Assim, o presente trabalho baseou-se na otimização da formulação de sistemas de entrega baseados no polímero de quitosano, tendo sido explorados quatro tipos de quitosano com diferentes pesos moleculares: quitosano de alto peso molecular (HMW, 200-500 kDa), baixo peso molecular (LMW, 50-190 kDa), 20 kDa e 5 kDa. A técnica utilizada para formular os sistemas foi a gelificação ionotrópica, baseada em ionic crosslinking. A formulação dos sistemas ocorreu pela interação entre os polímeros de quitosano, que apresentam carga positiva, o crosslinker tripolifosfato de sódio (TPP) e o ADN plasmídico (pADN), codificando E7 mutado, que apresentam carga negativa. A nclusão do TPP teve como objetivo compactar as nanopartículas formadas e aumentar a sua estabilidade, conferindo uma forma esférica mais definida. Para conseguir otimizar a formulação dos sistemas de entrega com cada polímero explorado, foi utilizada uma ferramenta de desenho de experiências (DoE). Esta ferramenta permitiu combinar vários parâmetros/inputs em simultâneo (concentrações de quitosano e TPP) para obter as condições ótimas de formulação dos sistemas com base nas respostas escolhidas (tamanho, índice de polidispersividade (PDI) e potencial zeta), de modo a realizar o mínimo número de experiências possível. Após a realização dos ensaios propostos e caracterização das propriedades das formulações obtidas, foram introduzidas as respetivas respostas no programa de DoE e realizada uma análise estatística. Os modelos lineares e quadráticos obtidos foram estatisticamente significativos (p-valor <0,05) e o “lack of fit” não significativo, com coeficiente de determinação adequado. Todos os pontos ótimos previstos para cada polímero de quitosano foram validados com sucesso. Posteriormente, outros estudos foram conduzidos para avaliar as propriedades dos sistemas de entrega formulados com as condições definidas nos pontos ótimos. Foram realizadas análises de microscopia eletrónica de varrimento (SEM) para avaliar a morfologia, espectroscopia de infravermelhos da transformada por Fourier para avaliar as propriedades químicas e grupos funcionais específicos, diferentes ensaios de estabilidade para avaliar a resistência e a libertação de ADN, e por último, ensaios de citotoxicidade para assegurar a biocompatibilidade das nanopartículas de quitosano. Os quatro sistemas de entrega desenvolvidos apresentavam características satisfatórias, sendo constituídos por nanopartículas esféricas ou ovais, as quais apresentavam boa estabilidade e resistência, retenção de ADN e boa biocompatibilidade. Neste sentido, a ferramenta DoE revelou ser uma ferramenta adequada para explorar e adaptar as características dos nanosistemas quitosano/TPP/pADN, contribuindo significativamente para desenvolver um sistema com propriedades adequadas de encapsulação, transporte e entrega de um pADN, proporcionando assim um avanço no campo de pesquisa da entrega de vacinas de ADN

Deep Cytometry: Deep learning with Real-time Inference in Cell Sorting and Flow Cytometry

Deep learning has achieved spectacular performance in image and speech recognition and synthesis. It outperforms other machine learning algorithms in problems where large amounts of data are available. In the area of measurement technology, instruments based on the photonic time stretch have established record real-time measurement throughput in spectroscopy, optical coherence tomography, and imaging flow cytometry. These extreme-throughput instruments generate approximately 1 Tbit/s of continuous measurement data and have led to the discovery of rare phenomena in nonlinear and complex systems as well as new types of biomedical instruments. Owing to the abundance of data they generate, time-stretch instruments are a natural fit to deep learning classification. Previously we had shown that high-throughput label-free cell classification with high accuracy can be achieved through a combination of time-stretch microscopy, image processing and feature extraction, followed by deep learning for finding cancer cells in the blood. Such a technology holds promise for early detection of primary cancer or metastasis. Here we describe a new deep learning pipeline, which entirely avoids the slow and computationally costly signal processing and feature extraction steps by a convolutional neural network that directly operates on the measured signals. The improvement in computational efficiency enables low-latency inference and makes this pipeline suitable for cell sorting via deep learning. Our neural network takes less than a few milliseconds to classify the cells, fast enough to provide a decision to a cell sorter for real-time separation of individual target cells. We demonstrate the applicability of our new method in the classification of OT-II white blood cells and SW-480 epithelial cancer cells with more than 95% accuracy in a label-free fashion

Gold Nanosystems Covered with Doxorubicin/DNA Complexes: A Therapeutic Target for Prostate and Liver Cancer

Different gold nanosystems covered with DNA and doxorubicin (Doxo) were designed and synthesized for cancer therapy, starting from Au@16-Ph-16 cationic nanoparticles and DNA–Doxo complexes prepared under saturation conditions. For the preparation of stable, biocompatible, and small-sized compacted Au@16-Ph-16/DNA–Doxo nanotransporters, the conditions for the DNA–Doxo compaction process induced by gold nanoparticles were first explored using fluorescence spectroscopy, circular dichroism and atomic force microscopy techniques. The reverse process, which is fundamental for Doxo liberation at the site of action, was found to occur at higher CAu@16-Ph-16 concentrations using these techniques. Zeta potential, dynamic light scattering and UV–visible spectroscopy reveal that the prepared compacted nanosystems are stable, highly charged and of adequate size for the effective delivery of Doxo to the cell. This fact is verified by in vitro biocompatibility and internalization studies using two prostate cancer-derived cell lines (LNCaP and DU145) and one hepatocellular carcinoma-derived cell line (SNU-387), as well as a non-tumor prostate (PNT2) cell line and a non-hepatocarcinoma hepatoblastoma cell line (Hep-G2) model used as a control in liver cells. However, the most outstanding results of this work are derived from the use of the CI+NI combined treatments which present strong action in cancer-derived cell lines, while a protective effect is observed in non-tumor cell lines. Hence, novel therapeutic targets based on gold nanoparticles denote high selectivity compared to conventional treatment based on free Doxo at the same concentration. The results obtained show the viability of both the proposed methodology for internalization of compacted nanocomplexes inside the cell and the effectiveness of the possible treatment and minimization of side effects in prostate and liver cancer

Modulating the catalytic activity of enzyme-like nanoparticles through their surface functionalization

The inclusion of transition metal catalysts into nanoparticle scaffolds permits the creation of catalytic nanosystems (nanozymes) able to imitate the behaviour of natural enzymes. Here we report the fabrication of a family of nanozymes comprised of bioorthogonal ruthenium catalysts inserted in the protective monolayer of gold nanoparticles. By introducing simple modifications to the functional groups at the surface of the nanozymes, we have demonstrated control over the kinetic mechanism of our system. Cationic nanozymes with hydrophobic surface functionalities tend to replicate the classical Michaelis Menten model, while those with polar groups display substrate inhibition behaviour, a key mechanism present in 20% of natural enzymes. The structural parameters described herein can be used for creating artificial nanosystems that mimic the complexity observed in cell machinery. © 2018 The Royal Society of Chemistry

Dynamic self-assembling DNA nanosystems: design and engineering

Over the last thirty years, DNA has proven to be a great candidate for engineering nanoscale architectures. These DNA nanostructures have been applied in areas such as single-molecular analyses, nanopatterning, diagnostics and therapeutics. One of the most commonly-used techniques to engineer DNA-based two- and three-dimensional functional nanostructures is DNA origami, wherein a long single-stranded DNA (called scaffold) is folded into a predetermined shape with the help of a set of shorter oligonucleotides (called staples). This thesis discusses a brief overview of DNA nanotechnology (design, assembly and applications) and three primary projects undertaken in the area of dynamic self-assembling DNA nanosystems: 1, a self-assembly design strategy that vastly expands the utility of DNA origami, 2, a DNA origami-based reconfigurable nanosystem with potential as a force/energy balance and diagnostic tool, and 3, a collaborative initiative on computational analyses and experimental verification for improving efficiency of DNA nanoengineering