Conventional computational methods available today for studying macromolecules and their complexes are limited to simulating short time frames and are insufficient to study processes of interest related to their function that usually occur In nature on longer time scales. Alternative methods that extend our capabilities continue to be proposed, and most often involve some kind of reduction In complexity or representation in order to simulate these biological processes on longer time and length scales. The ability to investigate through simulation the structural and functional properties of protein macromolecular complexes IS of particular importance in the field of bionanotechnology, whose goal IS to harness nanoscale devices made from or inspired by biological counterparts. Clearly then, methods are needed that can capture the large changes seen In macromoleculat assemblies to elucidate important principles of their structure and function and apply these to the nanomachines envisaged in bionanotechnology. At the forefront of this field lie the nanomotors, whose biological counterparts, the molecular protein motors, are used in cells to drive a host of essential processes with an amazing degree of efficiency and precision. The work in this thesis describes the development of a hierarchic modeling paradigm applied toward simulating the processi ve movement of the molecular myos m motor protein along an actin filament track. In the hierarchic model, three different levels of protein structure resolution are represented, with the level of detail changing according to the degree of interaction among the molecules, the integrity of which is maintained using a tree of spatially organized bounding volumes. Although applied to an acto-myosin system, the hierarchic framework is general enough so that it may easily be adapted to a number of other biomolecular systems of interest within the bionanotechnology field.

<p>  </p> <p>Conventional clinical therapies for head and neck squamous cell carcinomas (HNSCC) include surgical resection, chemotherapy (CT), and radiotherapy (RT). For locally advanced HNSCC, the CT-RT combination (“chemoradiation”) has been shown to be more effective than CT or RT alone and is the current standard of care. A novel radiation-controlled drug release nanoparticle formulation has been developed to realize the maximum benefits of intratumoral CT-RT. Under the application of low-dose X-ray, the formulation is capable of releasing PTX and providing localized, controlled, and sustained drug release for improvements in therapeutic efficacies.</p>

<p>Drug-delivery nano-systems enhances the potency of bioactive molecules due to its increase membrane permeability, as a result of their sub-cellular size. The concept of engineered nano-carriers may be a promising route to address confounding challenges in agriculture that could lead to an increase in crop production while reducing the environmental impact associated with crop protection and food production. A key motivation of this work is to evaluate the potential use of drug delivery nanosystems in agriculture, especially in the area of disease control. To this end, identifying the most suitable materials to serve as carrier and cargo is imperative. Understanding their bioactive properties and their physical-chemical characteristics is critical because these influences not only their biological effects on plants and environmental impact, but also, the fabrication process and potential scaling-up, enabling practical and relevant field applications in the future. </p> <p> </p> <p>In this work, chitosan was selected as nano-carrier material because of its biological and chemical properties. The chemical structure of chitosan allows spontaneous assemble of core-shell like nanostructures via ionic gelation, has enabled it to be used as nano-carrier biomaterial intended for delivery of bioactive cargo. In agriculture, the use of chitosan is of special interest due to its immune-modulatory activity elicited in plants. However, due to its inherent molecular heterogenicity, the formulation and fabrication of stable and low inter-batch variability chitosan nanocarriers via ionic gelation is difficult and time consuming.</p> <p> </p> <p>A myriad of different bioactive molecules has been tested as payload, encapsulated into chitosan-based delivery nano-systems for a range of purposes ranging from biomedicine, pharmaceutical, food and agriculture. In this work plant derived essential oils were selected as bioactive payload. Essential oils are at the core of the plant communication process with their phytobiome, including plant pathogens. Molecules from essential oils can carry an air-borne message serving as a plant-to-plant communication system (a phenomenon known as allelopathy) that activate the plant defense mechanisms. Encapsulation of essential oils into chitosan nanocarriers is only possible by forming nano-emulsions. </p> <p>Despite the potential benefits from the use of chitosan and essential oils in agriculture, its use at a large scale has been hindered by the overwhelming inconsistencies in the current literature, regarding their formulation and fabrication. This work addresses these problems and presents evidence that support the feasibility of producing highly chitosan nanocarriers loaded with essential oils, in a facile and rapid way, using FDA-grade materials only, without the need of expensive or specialized instrumentation. </p> <p> </p> <p>The plant-pathogen compatible interaction between <i>A. thaliana</i> and <i>B. cinerea</i> was used as biological model to test the hypothesis that chitosan nano-carriers and essential oil nano-emulsions can enhance the quantitative disease resistance of plants against broad host-range necrotrophic pathogens. We found that these treatments display a dose-dependent response in plants triggering a systemic immune response. Image-based phenotyping analysis showed that chitosan nanoparticles alone, as well as loaded with d-limonene, significantly enhanced the disease resistance of <i>A. thaliana</i> against <i>B. cinerea</i>. Nano-emulsions using essential oils from cinnamon, clove, coriander and red thyme also produced similar effects on the defense response in the pathosystem under study. Functional analysis of the differentially expressed genes from treated plants revealed that these treatments up-regulated the biological process involved in “stress management”, while down-regulated the biological process required for normal growth and development during ideal, non-stressful conditions.</p>

No / The most important issue during synthesis of nanoparticles (NPs) is to avoid particle agglomeration and adhesion. There have been several attempts to use special substances such as organic surfactants, polymers and stable ligands for this purpose. In this study, silver NPs were synthesised with and without gelatin macromolecules, as a green natural biopolymer, which resulted in NPs with varying shapes and sizes. The effect of morphological characteristics on the antibacterial and antifungal properties of the synthesised NPs were studied, by comparing Gram-negative (Escherichia coli) versus Gram-positive (Staphylococcus aureus) bacteria as well as fungi (Candida albicans) by calculation of minimal inhibition concentration value. The results corresponded well with the assumptions on the effects of shape and size on the antibacterial and antifungal properties of the studied NPs.

This thesis describes methods for constructing nanopatterned surfaces to array DNA. These surfaces enable direct observation of heretofore-unseen single-molecule reactions, eliminating bulk effects and enabling scientists to examine DNA mismatch repair and replication, including the first direct visualization of proteins binding to a target mismatch. This also facilitates directed self-organization of nanoscale features on a patterned substrate using DNA as an assembly tool. To make techniques for single-molecule visualization of biological processes more accessible, we have developed a novel technology called "DNA curtains," in which a combination of fluid lipid bilayers, nanofabricated barriers to lipid diffusion, and hydrodynamic flow can organize lipid-tethered DNA molecules into dened patterns on the surface of a microfluidic sample chamber. Using DNA curtains, aligned DNA molecules can be visualized by total internal reflection fluorescence microscopy, allowing simultaneous observation of hundreds of individual molecules within a field-of-view. Ultimately, this results in a 100X improvement in experimental throughput, and a corresponding increase in statistically signicant amounts of data. We also demonstrate site-specific labeling of DNA using DNA analogues, such as peptide nucleic acid (PNA), locked nucleic acid (LNA), and techniques such as nick-translation. Through PNA invasion, labeled DNA was self-assembled in arrays on surfaces and tagged with gold nanoparticles. In this work, DNA formed a template to self-assemble a nanoparticle in between nanoimprinted AuPd dots. Surface-based self-assembly methods offer potential for DNA employment in bottom-up construction of nanoscale arrays. This offers further proof that DNA can be useful in directed self-assembly of nanoscale architectures.

Nanotechnology-based solutions have gained burgeoning attention in medical research, as compared with conventional therapeutic modalities, they offer advantages in efficacy, safety, and scalability. Researchers have been developing fluidic systems for nanoformulations over recent decades. Despite promising results, the clinical potential of the current nanosystems is still limited by insufficient cargo (drug and gene) loading, low production, high toxicity, low colloidal stability, unsatisfied bioavailability, and batch-to-batch variation. Flash-based self-assembly is a recently developed technology that can manufacture nanoformulations in facile, consistent, reproducible, and scalable manners. Due to the turbulent and dynamic flow generated in the mixing chamber, biomaterials self-assemble into uniform nanoparticles (NPs) through precipitation or complexation. We modified and manufactured a number of flash-based systems and evaluated their dynamic mixing profiles through simulation and empirical testing for polyplex formation and nanoparticle coating, as the dynamic fluidic control is the key for biomaterial complexation and nanoparticle coating, which provides better nanoparticle colloidal stability. In Chapter 2, we formulated polyplexes and lipid-coated NPs with controllable size and enhanced colloidal stability by exploiting the dynamic mixing of the flash-based system. Bio-inspired nanosystems with engineered functions have been advancing the field of nanomedicine. Incorporating bio-inspired components can provide nanosystems with productive ways of interacting with their surroundings by diminishing nonspecific interactions or enhancing specific targeting. Membranes from different cell types, and even organisms, can be employed and merged to meet specific goals. We derived cell membranes from distinctive mammalian cell lines to improve nanosystems with smart biological interactions, such as preserving neo-antigens or enhancing specific targeting. Another potent property of utilizing cell membranes is that they provide NPs with colloidal stability. Recent studies have reported the use of cell membrane coating onto NPs in drug delivery, imaging, phototherapies, and detoxification. The derived components from the original cell source bestow the NPs with their inherent functionality without additional complicated modulation. Cell membrane coating is a top-down technique that directly derives and harnesses the natural components, evading the technical and procedural challenges in bottom-up fabrication. However, current membrane coating techniques have problems of batch-to-batch variation and low production yield, which limits its potential for clinical translation. Taking advantage of flash-based self-assembly, we standardized and scaled up the cell membrane-coating process, which is difficult to achieve in bulk mixing approaches. The optimization of cell membrane coating was explored using various simulations. The time and cost for experimental design and optimization were reduced considerably. Cell membranes derived from tumor cells contain a rich source of tumor antigens. With the potential of cell membrane coating using flash-based self-assembly, we applied the produced cell-membrane-coated mesoporous silica nanoparticles (MSN) as a biomimetic nanovaccine for cancer immunotherapy in Chapter 3. Oral delivery of drugs and genes is a relatively convenient, patient-friendly, and safe approach. Targeted and controlled oral delivery of active pharmaceutical ingredients (API) of biomimetic nanocarriers offers significant advantages in efficacy and safety compared to conventional modalities. Besides mammalian cells, the unique functionalities of other prokaryotic and eukaryotic cell types, such as bacterium and yeasts, were exploited for macromolecule delivery. Baker’s yeast, a common yeast strain closely associated with food preparation, contains valuable polysaccharides that were reported to specifically bind to the dectin-1 receptors on the specialized intestinal epithelial cells and monocytes. Exploiting the yeast’s cell wall is a biomimetic strategy when designing an oral carrier for targeted oral drug and gene delivery. We demonstrated that the specific recognition between the microfold cells (M-cells) of the small intestine and the polysaccharides on the yeast cell wall enhances the transport of yeast-based formulations across the gut epithelium and into the lymphatic tissues in chapter 4. Utilizing the micron-sized yeast capsule or decorating a nanoparticle surface with processed yeast cell wall fragments, therapeutics were efficiently delivered to the target site through the oral path. The yeast-based formulations are biomimetic systems for targeted oral delivery of therapeutics. Taken together, the goal of this thesis is to close the gap between laboratory research and clinical translation by exploring the versatility and robustness of the developed flash technology, exploiting flash-based self-assembly for scalable production of the lipid and cell-membrane-coated nanosystems, and developing a relatively safe yeast-based drug and gene delivery platform.

Détecter un ensemble de marqueurs biologiques dans un sérum de patient ou bien des molécules spécifiques d'un herbicide dans un échantillon prélevé dans l'eau d'une rivière ? Etre capable de transformer une interaction biologique en un signal électrique ou encore déposer des volumes infiniment faibles de molécules biologiques sur une surface solide à des fins de diagnostique ? Passer de la fabrication de microcapteurs inertiels pour la navigation à la conception et au développement de biocapteurs micromécaniques ? Nous démontrons que le fil conducteur permettant de faire le lien entre ces domaines en apparence disjoints est matérialisé par des micro- et nanosystèmes électromécaniques développés au sein du LAAS à partir de la feuille blanche jusqu'à l'intégration du système avec son électronique associée. Quel lendemain pour les bio- microsystèmes électromécaniques ? Faut-il encore miniaturiser ? Est-il pertinent d'entreprendre le contraire ? Comment poursuivre l'aventure transdisciplinaire en étant sûr du fait que la réussite est au bout de la route ? Nous tentons de répondre à l'ensemble de ces questions tout au long de ce manuscrit retraçant l'ensemble de nos travaux de recherche effectués au LAAS et ailleurs depuis l'an 2000.

<p>I. Gallium-hemoglobin Coated Silver Nanoparticles for Antimicrobial Photodynamic Therapy Against Bacterial Pathogens One of the mechanisms for bacterial pathogens’ hemin acquisition is through cell-surface hemin receptors (CSHRs), which are responsible for rapid hemin recognition. GaPpIX, as a hemin analog, can be rapidly taken up by CSHR-expressing bacteria, such as <i>Staphylococcus aureus</i> (<i>S.aureus)</i>. Previous works shown that GaPpIX has aPDI activity at micromolar level of concentration following 10 seconds of 405-nm light exposure using LED array. The photosensitizing ability of GaPpIX can be further enhanced by incorporating with hemoglobin (GaHb) and 10 nm silver nanoparticles (AgNP). The results suggested a higher aPDI activity of GaHb-AgNP than any of its components against MRSA strains and neglectable cytotoxicity against keratinocytes. GaHb-AgNPs were also found having aPDI activity against intracellular MRSA and <i>Mycobacterium abscessus </i>but not effective against <i>S. aureus</i> biofilm. GaHb-AgNPs have no significant toxicity toward macrophages with concentrations lower than 22.64 μg/mL.</p> <p> </p> <p>II. Synthesis and Properties of Magnetic Gold Nanoparticles</p> <p> Superparamagnetic gold nanoparticles support hybrid magnetic and plasmonic properties that can be exploited for a variety of applications. In this paper we present new insights on the synthesis of magnetic gold nanoparticles (MGNPs) with an emphasis on efficiency, scalability, and waste reduction, supported by a comprehensive analysis of their physical and materials properties. Aqueous suspensions of colloidal Fe₃O₄ are conditioned with 5-kDa polyethylene glycol and L-histidine to mediate the nucleation and growth of gold by a mild reducing agent. Isotropic MGNPs on the order of 100 nm can be synthesized using scalable reaction conditions with Au:Fe mole ratios as low as 1:2 and cleansed with generally regarded as safe (GRAS) chemicals for the removal of residual iron oxide. High-resolution energy-dispersive x-ray imaging of individual MGNCs revealed these to be ultrafine composites of gold and SPIO rather than core–shell structures. The attenuated total reflectance infrared (ATR-IR) spectroscopy and Raman spectroscopy indicated that the cleansing step does change the optical properties of the synthesized MGNPs. Magnetometry of MGNCs in bulk powder form confirmed their superparamagnetic nature, with bulk moments between 6 to 7 emu/g.</p>

Advances in recent nanoscience technologies have generated a new compilation of biocompatible, fluorescent nanoparticles derived from semiconductor quantum dots (QDs). QDs are extremely small in size and possess very large surface areas, which gives them unique physical properties and applications distinct from those of bulk systems. When exposed to biological fluid, these QDs may become coated with proteins and other biomolecules given their dynamic nature. These protein-QD systems may affect or enhance the changes in protein structure and stability, leading to the destruction of biological function. It is believed that these QDs can act as nucleation centers and subsequently promote protein fibril formation. Protein fibrillation is closely associated with many fatal human diseases, including neurodegenerative diseases and a variety of systemic amyloidoses. This topic of protein-QD interaction brings about many key issues and concerns, especially with respect to the potential risks to human health and the environment. Herein, the behavioral effects of dihydrolipoic acid (DHLA)-capped CdSe/ZnS (core/shell) QDs in hen egg-white lysozyme (HEWL) and human serum albumin (HSA) protein systems were systematically analyzed. This study gives rise to a better understanding of the potentially useful application of these protein-QD systems in nanobiotechnology and nanomedicine as a bioimaging tool and/or as a reference for controlled biological self-assembly processes.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2018-05-21T15:25:31Z No. of bitstreams: 1 PDF - Anna Emmanuela Medeiros de Brito.pdf: 29262242 bytes, checksum: b0fe663b1a5e1b8e8d845a03a6b68c4a (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2018-05-23T16:59:52Z (GMT) No. of bitstreams: 1 PDF - Anna Emmanuela Medeiros de Brito.pdf: 29262242 bytes, checksum: b0fe663b1a5e1b8e8d845a03a6b68c4a (MD5) / Made available in DSpace on 2018-05-23T16:59:52Z (GMT). No. of bitstreams: 1 PDF - Anna Emmanuela Medeiros de Brito.pdf: 29262242 bytes, checksum: b0fe663b1a5e1b8e8d845a03a6b68c4a (MD5) Previous issue date: 2017-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq / Acute lymphocytic leukemia (ALL) is a type of cancer that compromises the maturation of blood cells of the lymphoblastic lineage, being prevalent in children, but with a good chance of cure due to chemotherapy. The biopharmaceutical L-asparaginase (L- ASNase) is one of the main drugs used in the treatment of this neoplasm, but the immunogenicity derived from the bacterial origin of the enzyme currently produced and the consequent short half-life are challenges to be overcome by the pharmaceutical industry. In this sense, nanobiotechnology is a broad platform for the development of drug delivery aimed at the transport of therapeutic enzymes, besides being able to overcome these problems, it still allows better protein stability with regard to aggregation and denaturation that result in a decrease in enzymatic activity and consequently the pharmacological action. Thus, the objective of this work was to obtain and characterize poly (lactic acid-co-glycolic acid) nanoparticles (PLGA) for L-ASNase encapsulation. The double emulsification method by homturrizing with ultraturrax or cavitation with ultrasound probe was used to obtain the systems, using different times (30 or 60 s) and from two types of PLGA 50:50 with different molecular weights (30-60 KDa e 24-38 KDa) and polyvinyl alcohol (PVA) at concentrations of 0.5, 1, 1.5 and 2%. The size and polydispersity of the nanoparticles were evaluated by dynamic light scattering (DLS). The evaluation of the ASNase encapsulation was performed by quantification of total proteins by the indirect (in the supernatant) and direct method (in the ruptured nanoparticle system). By the nessler method, it was possible to observe that the encapsulated enzyme presented greater activity than the free enzyme. The enzyme release study was performed by dialysis, where < 60% of protein was released in 48 hours. The L-ASNase encapsulation monitoring was performed using native gel electrophoresis and zymogram. Circular dichroism was also used to evaluate the conformational changes of the encapsulated enzyme. From PLGA 30-60 KDa systems were obtained with sizes predominantly between 400 and up to more than 1 μm, with large variation in sizes between them, and for PLGA 24-38 KDa the size range is from 380 nm to 670 nm. Cavitation and higher concentration of PVA resulted in the formation of systems without coalescence. The system made with PLGA 24-38 KDa obtained by cavitation with 1% PVA with homogenization time of 60 s was chosen for ASNase encapsulation and presented encapsulation efficiency by the direct method of 86.67% (± 1.84) and by the method of 95.35% (± 0.06). According to the hemolysis essay, the systems with and without the enzyme were nonhemolytic. / A Leucemia Linfoide Aguda (LLA) é um tipo de câncer que compromete a maturação das células sanguíneas da linhagem linfoblástica, sendo prevalente em crianças, mas com boas chances de cura advinda da quimioterapia. O biofármaco L-asparaginase (L- ASNase) é um dos principais fármacos usados no tratamento desta neoplasia, porém a imunogenicidade advinda da origem bacteriana da enzima atualmente produzida e o consequente tempo de meia-vida curto são desafios a serem vencidos pela indústria farmacêutica. Neste sentido, a nanobiotecnologia é uma ampla plataforma para o desenvolvimento de drug delivery visando o carreamento de enzimas terapêuticas, podendo além de contornar estes problemas, ainda permitir melhor estabilidade das proteínas no que diz respeito à agregação e desnaturação que resultam em diminuição da atividade enzimática e consequentemente da ação farmacológica. Assim, o objetivo deste trabalho foi a obtenção e caracterização de nanoparticulas do tipo nanoesferas de poli (ácido lático- co- glicólico) (PLGA) para encapsulação da L-ASNase. O método de dupla emulsificação por homogeinização com ultraturrax ou cavitação com sonda de ultrassom foi usado para obtenção dos sistemas, empregando diferentes tempos (30 ou 60 s) e a partir de dois tipos de PLGA 50:50 com diferentes pesos moleculares (30-60 KDa e 24-38 KDa) e álcool polivinílico (PVA) nas concentrações de 0,5;1;1,5 e 2%. O tamanho e polidispersão das nanopartículas foram avaliados por espalhamento de luz dinâmico (DLS). A avaliação da encapsulação da L-ASNase foi realizada por meio da quantificação de proteínas totais através do método indireto (no sobrenadante) e direto (no sistema de nanapartículas rompido). Através da nesslerização foi possível observar que a enzima encapsulada apresentou maior atividade que a enzima livre. O estudo de liberação da enzima foi realizado por meio de diálise, onde foi liberada < 60% de proteína em 48 horas. O monitoramento da encapasulação da L-ASNase foi feito por meio de eletroforese com gel nativo e zimograma. Também foi empregado dicroísmo circular para avaliação das alterações conformacionais da enzima encapsulada. A partir do PLGA 30-60 KDa foram obtidos sistemas com tamanhos predominantemente entre 400 nm e até mais de 1 µm, com grande variação de tamanhos entre eles, já para PLGA 24-38 KDa a faixa de tamanho é de 380 nm a 670 nm. A cavitação e maior concentração de PVA resultou na formação de sistemas sem coalescência. O sistema feito com PLGA 24-38 KDa obtido por cavitação com PVA 1% com tempo de homogeneização de 60 s foi escolhido para encapsulação da ASNase e apresentou eficiência de encapsulação pelo método direto de 86,67 % (± 1,84) e pelo método indireto de 95,35%(± 0,06). De acordo com o ensaio de hemólise, os sistemas com e sem a enzima se mostraram não hemolíticos.