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Patricia Brassolatti,1 Joice Margareth de Almeida Rodolpho,1 Krissia Franco de Godoy,1 Cynthia Aparecida de Castro,1 Genoveva Lourdes Flores Luna,1 Bruna Dias de Lima Fragelli,1 Matheus Pedrino,1 Marcelo Assis,2 Marcel Nani Leite,3 Juliana Cancino-Bernardi,4 Carlos Speglich,5 Marco Andrey Frade,3 Fernanda de Freitas Anibal1 1Laboratory of Inflammation and Infectious Diseases, Department of Morphology and Pathology, Federal University of São Carlos, São Carlos, São Paulo, Brazil; 2Center for the Development of Functional Materials, Department of Chemistry, Federal University of São Carlos, São Carlos, São Paulo, Brazil; 3Division of Dermatology - Wound Healing & Hansen’s Disease Lab, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil; 4Nanomedicine and Nanotoxicology Group, Physics Institute of São Carlos, University of São Paulo, São Carlos, São Paulo, Brazil; 5Leopoldo Américo Miguez de Mello CENPES/Petrobras Research Center, Rio de Janeiro, Rio de Janeiro, BrazilCorrespondence: Patricia Brassolatti, Departamento de Morfologia e Patologia UFSCar, Rod. Washington Luís, Km 235 Caixa Postal 676, São Carlos, CEP. 13565-905, SP, Brazil, Tel +551633518325, Fax +551633518326, Email patty.brassolatti@gmail.comPurpose: Nanoparticles are resources of advanced nanotechnology being present in several products. Titanium dioxide nanoparticles are among the five most widely used NP currently expanding their benefits from the oil industry to the areas of diagnostic medicine due to their properties and small size. However, its impact on human health is still controversial in the literature. We aimed to evaluate the cytotoxicity of a new titanium NP functionalized with sodium carboxylic ligand (COOH−Na+) in human keratinocytes (HaCaT) and human fibroblasts (HDFn).Methods: The physical-chemical characterization was performed by the transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential techniques, respectively. MTT and LDH assays were used to assess cytotoxicity and cell membrane damage respectively, ELISA to identify the inflammatory profile and, reactive oxygen species assay and cytometry to detect reactive oxygen species and their relationship with apoptosis/necrosis mechanisms.Results: The results demonstrated a decrease in cell viability at the highest concentrations tested for both cell lines, but no change in LDH release was detected for the HaCaT. The cell membrane damage was found only at 100.0 μg/mL for the HDFn. It was demonstrated that cytotoxicity in the highest concentrations evaluated for both cell lines for the 72 h period. The HDFn showed damage to the cell membrane at a concentration of 100 μg/mL followed by a significant increase in reactive oxygen species production. No inflammatory profile was detected. The HaCaT showed apoptosis when exposed to the highest concentration evaluated and HDFn showed both apoptosis and necrosis for the same concentration.Conclusion: Thus, it is possible to conclude that the cytotoxicity mechanism differs according to the cell type evaluated, with HDFn being the most sensitive line in this case, and this mechanism can be defined in a dose and time dependent manner, since the highest concentrations also triggered death cell.Keywords: nanoparticle, titanium, cytotoxicity, human skin cells

Extracellular vesicles (EVs) and cell membrane nanoghosts are excellent coatings for nanomaterials, providing enhanced delivery in the target sites and evasion of the immune system. These cell-derived coatings allow the exploration of the delivery properties of the nanoparticles without stimulation of the immune system. Despite the advances reported on the use of EVs and cell-membrane coatings for nanomedicine applications, there are no standards to compare the benefits and main differences between these technologies. Here we investigated macrophage-derived EVs and cell membranes-coated gold nanorods and compared both systems in terms of target delivery in cancer and stromal cells. Our results reveal a higher tendency of EV-coated nanorods to interact with macrophages yet both EV and cell membrane-coated nanorods were internalized in the metastatic breast cancer cells. The main differences between these nanoparticles are related to the presence or absence of CD47 in the coating material, not usually addressed in EVs characterization. Our findings highlight important delivery differences exhibited by EVs- or cell membranes- coated nanorods which understanding may be important to the design and development of theragnostic nanomaterials using these coatings for target delivery.

This study offers a novel oral pregabalin (PG)-loaded drug delivery system based on chitosan and hypromellose phthalate-based polymeric nanocomposite in order to treat neuropathic pain (PG-PN). PG-PN has a particle size of 432 ± 20 nm, a polydispersity index of 0.238 ± 0.001, a zeta potential of +19.0 ± 0.9 mV, a pH of 5.7 ± 0.06, and a spherical shape. Thermal and infrared spectroscopy confirmed nanocomposite generation. PG-PN pharmacokinetics was studied after a single oral dose in male Wistar rats. PG-PN showed greater distribution and clearance than free PG. The antinociceptive effect of PG-PN in neuropathic pain rats was tested by using the chronic constriction injury model. The parameter investigated was the mechanical nociceptive threshold measured by the von Frey filaments test, PG-PN showed a longer antinociceptive effect than free PG. The rota-rod and barbiturate sleep induction procedures were used to determine adverse effects, the criteria included motor deficit and sedative effects. PG-PN and free PG had plenty of motors. PG-PN exhibited a less sedative effect than free PG. By prolonging the antinociceptive effect and decreasing the unfavorable effects, polymeric nanocomposites with pregabalin have shown promise in treating neuropathic pain.

This study aimed the enzymatic decyl esters production by hydroesterification, a two-step process consisting of hydrolysis of refined soybean (RSBO) or used soybean cooking (USCO) oils to produce free fatty acids (FFA) and further esterification of purified FFA. Using free lipase from Candida rugosa (CRL), about 98% hydrolyses for both oils have been observed after 180 min of reaction using a CRL loading of 50 U g

Branched anisotropic gold nanostructures present distinguished performance acting both as contrast agents for photoacoustic imaging and as active agents for photothermal therapies. Despite advances in their fabrication methods, the synthesis of such gold nanomaterials in a simple and reproducible way is still a challenge. In this paper, we report the development of branched anisotropic gold nanoparticles, the so-called gold nanoflowers (AuNFs), as near-infrared active theragnostic materials for cancer therapy and diagnosis. In situ chemical synthesis of the AuNFs was optimized to obtain monodisperse nanoflowers with controllable size and optical properties. Upon varying the temperature and gold ion concentrations, it was possible to tune the optical activity of the nanoparticles from 590 to 960 nm. The AuNFs exhibited good stability in the cell culture medium, and under laser irradiation. Photoacoustic imaging revealed that the NFs could be imaged in phantom systems even at low concentrations. In vitro tests revealed that the nanoflowers were effective in the photothermal therapy of a rat hepatocarcinoma (HTC) cell lineage. In addition, no toxicity was observed to mouse fibroblast (FC3H) cells incubated with the AuNFs. Our results reveal a simple method to synthesize branched anisotropic gold nanostructures, which is a promising platform for photothermal and photoacoustic therapies.

Nanomedicine is a research area at the interface between nanotechnology and biotechnology that aims at developing nanosystems for diagnosis and therapy. As active materials to be used in both diagnosis and therapy, inorganic nanoparticles (INPs), in particular, are of great interest in nanomedicine. INPs have been applied in several therapy strategies, including active drug delivery, hyperthermia, and magnetothermia against cancer, and their use offer advantages in medical applications due to their physicochemical characteristics, such as localized surface plasmon resonance (LSPR) effect—present in Au and Ag nanoparticles, for example—and the magnetic and photoluminescence properties of magnetic nanoparticles and quantum dots, respectively. The application of INPs in molecular imaging and drug delivery has benefited from their reduced size, which allows the INPs to overcome cell barriers that conventional drugs cannot do, such as the blood–brain barrier. In addition to the physical and chemical properties of the INPs, their surface modifications using biomolecules provide specificity to the nanocarrier systems to increase absorption and biodistribution. This chapter describes the main characteristics and the most relevant applications of gold, silver, quantum dots, ceramics, and magnetic nanoparticles in biomedicine.

The search for new nanomaterials has brought to the multifactorial industry several opportunities for use and applications for existing materials. Carbon nanotubes (CNT), for example, present excellent properties which allow us to assume a series of applications, however there is concern in the industrial scope about possible adverse health effects related to constant exposure for inhalation or direct skin contact. Thus, using cell models is the fastest and safest way to assess the effects of a new material. The aim of this study was to investigate the cytotoxic profile in LA9 murine fibroblast lineage, of a new multi-walled carbon nanotube (MWCNT) that was functionalized with tetraethylenepentamine (TEPA) to obtain better physical-chemical characteristics for industrial use. The modifications presented in the CNT cause concern, as they can change its initial characteristics, making this nanomaterial harmful. HR-TEM, FE-SEM and zeta potential were used for the characterization. Cytotoxicity and cell proliferation tests, oxidative and nitrosative stress analyzes and inflammatory cytokine assay (TNF-α) were performed. The main findings demonstrated a reduction in cell viability, increased release of intracellular ROS, accompanied by an increase in TNF-α, indicating an important inflammatory profile. Confirmation of the data was performed by flow cytometry and ImageXpress with apoptosis/necrosis markers. These data provide initial evidence that OCNT-TEPA has a cytotoxic profile dependent on the concentration of LA9 fibroblasts, since there was an increase in free radicals, inflammation induction and cell death, suggesting that continuous exposure to this nanoparticle can cause damage to different tissues in the organism.

An electrochemical immunosensor was developed for the determination of apo-Tf (non-iron-bound) and holo-Tf (iron-bound) using polyclonal antibody transferrin (anti-Tf) immobilized at an electrode surface as a biorecognition platform. The monitoring was based on the anti-Tf binding with both Tf forms which allows the detection of cancer cells due to the constant iron cycle and the overexpression of anti-Tf on the cancer cell surface. The immunosensor characterization was performed using electrochemical impedance spectroscopy (EIS), which evaluated the impedimetric biorecognition of the antigens-antibody by the use of K4Fe(CN)6 redox group. The immunosensor was able to detect both forms of Tf in terms of charge transfer resistance (Rct). Analytical curves showed a limit of detection of 0.049 and 0.053 ng mL−1 for apo-Tf and holo-Tf, respectively. The immunosensor was applied to the detection of the two cancer cells A549 (lung carcinoma) and MCF-7 (breast carcinoma) and compared with BHK570, a healthy cell line. The impedimetric response of healthy cells differs significantly from that of the cancerous cells, as revealed by a Dunnett’s test in 95% confidence level—ca. 102 cells mL−1—indicating the feasibility of the immunosensor to discriminate both types of cells. The indirect detection of anti-Tf based on apo-Tf and holo-Tf binding can be considered an advanced approach for cancer recognition.

The understanding of the interactions between biomolecules and nanomaterials is of great importance in many areas of nanomedicine and bioapplications. Numerous studies in this area have been performed. However, toxicological aspects involving the interaction between phospholipids and carbon nanotubes (CNTs) remain undefined, especially for those cases in which a protein corona is not formed around the nanomaterial (corona-free nanomaterials). This study focuses on the interaction of Langmuir films of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC) with corona-free, single-walled CNTs. Surface pressure-area isotherms and sum-frequency generation (SFG) vibrational spectroscopy, a non-linear optical technique used to study surfaces and interfaces, were used to investigate the lipid tail orientation and conformation, aiming to understand the interactions between phospholipids and single walled carbon nanotubes functionalized by carboxylic acid (SWCNTs-COOH) at the air-water interface under low ionic strength conditions. Data from isotherms and SFG spectra revealed that the SWCNT adsorption at the air-water interface is induced by the presence of both lipids, although at a lesser extent for DPPG due to its anionic head group, which could result in repulsion of SWCNTs-COOH that also bear a negative charge. Furthermore, lipid monolayers remained conformationally ordered, indicating insertion of SWCNTs into the lipid monolayer. Our results corroborate previous works and simulations in the literature, but made it possible to perform an in-depth investigation of the interaction of these nanomaterials with components of phospholipid membranes.

Essa tese de doutoramento utiliza a auto-organização dos filmes finos layer-by-layer (LbL), auto-organização por alcanotióis mistas (SAMmix) e monocamada de Langmuir no desenvolvimento de dispositivos e novas metodologias para aplicações em nanomedicina. Foram desenvolvidos e aplicados biossensores utilizando as técnicas de LbL e SAM. Dentre os biossensores construídos está o sensor para óxido nítrico (NO•), que é de grande importância no sistema fisiológico. O sensor foi construído por meio da modificação de ultramicroeletrodos de fibra de carbono pela técnica LbL. A caracterização do sensor foi realizada por voltametrias e espectroscopias de impedância eletroquímica. Os resultados revelaram que a difusão de NO• é dependente do número de bicamadas empregadas e da disposição das moléculas no filme. O sensor com arquitetura CF-(PAMAM/NiTsPc), fibra de carbono (CF), ftalocianina de níquel tetrasulfonada (NiTsPc) e dendrímero poliamidoamina (PAMAM), apresentou o melhor sinal analítico. Além disso, foi analisada a detecção de NO• com interferentes como nitrito, nitrato, peróxido de hidrogênio, ácido ascórbico, dopamina, epinefrina e a norepinefrina. Os resultados mostraram alta seletividade devido à utilização do dendrímero PAMAM. O segundo biossensor utilizou a enzima acetilcolinesterase imobilizada em monocamadas auto-organizadas mistas (SAMmix) de alcanotióis. A detecção eletroquímica mostrou-se altamente sensível, uma vez que não há o uso do glutaraldeído como agente reticulante. Com essa plataforma foi possível desenvolver um biossensor de acetilcolina estável e robusto, sendo calculado o valor de Km app = 0,46x10-3 mol L-1, limite de detecção LD=3,32x10-10 mol L-1 e limite de quantificação LQ=1,11x10-9 mol L-1, valores inferiores aos encontrados na literatura, ressaltando a eficiencia da nova plataforma. Seguindo a mesma idéia de auto-organização, foram realizados estudos de nanotoxicidade utilizando modelos de membrana a partir de filmes de Langmuir. O principal objetivo foi elucidar a ação dos nanotubos de carbono (SWCNT), PAMAM e do nanocomplexo entre os dois materiais (SWCNT-PAMAM) nas membranas celulares, a nível molecular, usando um sistema modelo de membrana. A penetração de SWCNT e dos nanocomplexos em monocamadas lipídicas foi estudada utilizando microscopia de ângulo de Brewster (BAM) simultaneamente com cinética de absorção e pressão de superfície. Os resultados confirmaram a interação entre os nanomateriais e a membrana, indicando que a presença dos nanomateriais afeta o empacotamento dos lipídios. Foram realizados ainda estudos de citotoxicidade dos mesmos nanomateriais em sistemas celulares in vitro. Os resultados de citometria, proliferação celular, morfologia e inibição de adesão apresentaram-se evidenciaram que a combinação entre SWCNT e PAMAM, proporciona um maior índice de toxicidade em relação ao SWCNT, um comportamento diferente do que relatado nos componentes individuais. A toxicidade de nanocomplexos de SWCNT-PAMAM e de seus componentes individuais podem estar fortemente ligados ao tipo de material e como estes estão disponíveis no meio de cultura. Os estudos contidos nessa tese mostram a versatilidade dos filmes finos em sistemas auto-organizados e biomiméticos, e podem ser relevantes para o avanço de pesquisas sobre interação de nanomateriais e biossistemas. In this thesis we employed the concept of self-organization, including the layer-by-layer (LbL) technique, alkanethiols self-assembled monolayers (SAMmix) and Langmuir monolayers, to develop new methods for materials and devices manipulation for application in nanomedicine. Two different types of biosensors were developed. The first one was based on the LbL technique to detect nitric oxide (NO•), which is of great importance in the medicine. The second biosensor was based on SAM monolayers supporting acetylcholinesterase for pesticide monitoring. The NO• was constructed by modified carbon fiber (CF) assembled with nickel phtalocyanine tetrasulfonade (NiTsPc) and polyamidoamine dendrimer (PAMAM) in the form of ultramicroelectrodes (UMEs) by the LbL technique. The sensor was characterized using differential pulse voltammetry and electrochemical impedance spectroscopy. The results showed that NO• diffusion is dependent on the number of bilayers employed and the arrangement of molecules in the film. The sensor architecture with CF-(PAMAM/NiTsPc) presented the best analytical signal. In addition, we analyzed the detection of interfering with NO• as nitrite, nitrate, hydrogen peroxide, ascorbic acid, dopamine, epinephrine and norepinephrine. The results showed high selectivity due to the use of PAMAM dendrimer as selective layer. The second biosensor used the enzyme acetylcholinesterase immobilized on SAMmix. The electrochemical detection of carbaryl was highly sensitive, since there is no use of glutaraldehyde as crosslinking agent. Using acetylcholine as a probe, Kmapp value was determined at 0.46x10-3 mol L-1, with detection limit of 3.32x10-10 mol L-1 and quantification limit of 1.11x10-9 mol L-1, values lower than those found in the literature, highlighting the efficiency of the new platform. Langmuir films made of lipids were employed as cell membrane models, in order to investigate the interactions between single-wall carbon nanotubes (SWCNT), PAMAM and their nanocomplex (SWCNT-PAMAM) at the molecular level. The interation of SWCNT and nanocomplexes in lipid monolayers was studies using Brewster angle microscopy (BAM) in conjunction with absorption kinetics and surface pressure. The results confirmed the interaction between nanomaterials and the membrane, indicating that the presence of nanomaterials affects the packing of the lipids. Cytotoxicity studies were also employed to investigate the interaction of nanomaterials in in vitro cell systems. The results of flow cytometry, cell proliferation, morphology and inhibition of adhesion revealed the toxicological aspects of the materials, demonstrating a higher toxicity to the nanocomplex, compared to SWCNT, differently of the individual components. The toxicity of SWCNT nanocomplex and its individual components can be related to the type of material and how these materials are available in the culture medium. The studies in this thesis show the versatility of self-assembly thin films on biomimetic systems and may be relevant to the advance of research on the interaction of nanomaterials and biosystems.

Smart nanomaterials can selectively respond to a stimulus and consequently be activated in specific conditions, as a result of their interaction with electromagnetic radiation, biomolecules, or pH change. These nanomaterials are produced through distinct routes and can be used in artificial skin, drug delivery, and other biomedical applications. Here, we report on the fabrication of an antibacterial nanogel formed by aniline- and chitosan-containing silver nanoparticles (AgNp’s), with an average size of 78 ± 19 nm. The AgNp nanogel release was triggered by light at 405 nm. Specifically, the electronic energy vibration resulting from the interaction of the irradiation with the AgNp surface plasmon breaks the hydrogen bonds of the nanogels and releases AgNp’s. To understand the perturbation of AgNp-nanogels against bacteria, membrane model studies were performed using the main components of the cell membrane of Escherichia coli (E. coli), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) and 1,2...

Understanding the interactions occurring at the nano-bio interface is still a challenge. This study presents an investigation on the interactions between natural cell membranes and gold-based particles that were isolated from real - cancer and health - cells and reconstructed as monolayers. The influence of the morphology and surface charge of gold nanomaterials on the membrane structure, as well as the interface properties of the membranes was evaluated. Our results revealed that the interaction pathway of nanoparticles through the cell membrane was affected by the lipidic composition of each cell type evaluated. Moreover, we show that the surface charge of the nanoparticles plays a significant role in their ability to interact with cell membranes, as revealed by isothermal titration calorimetry, and atomic force microscopy analysis.

The increase of production and consumption of copper oxide nanostructures in several areas contributes to their release into aquatic ecosystems. Toxic effects of copper oxide nanoparticles (CuO NPs), in particular, on tropical aquatic organisms are still unknown, representing a risk for biota. In this study, the effects of rod-shaped CuO NPs on the Neotropical species Ceriodaphnia silvestrii and Hyphessobrycon eques were investigated. We also compared the toxicity of CuO NPs and CuCl2 on these species to investigate the contribution of particles and cupper ions to the CuO NPs toxicity. Considering the low copper ions release from CuO NPs (

Melatonin, the ‘chemical signal of darkness’, is responsible to regulate biological rhythms and different physiological processes. It is mainly produced by the pineal gland as a hormone in a rhythmic daily basis, but it may also be synthesized by other tissues, such as immune cells, under inflammatory conditions. Its abnormal circulating levels have been related to several diseases such as type 2 diabetes, Alzheimer’s disease and some types of cancer. Currently, melatonin is exclusively quantified by ELISA or radioimmunoassays, which although are very sensitive techniques and present low detection limits, usually require specialized personal and equipment, restricting the tests to a limited number of patients. To overcome such limitations, we developed a novel easy-to-use electrochemical immunosensor for rapid melatonin quantification. Anti-melatonin antibodies were immobilized into Indium tin oxide (ITO) platforms using (3-Aminopropyl)triethoxysilane (APTES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinkers. The platforms were assayed with synthetic and biologically-present melatonin containing samples. The developed device displayed a linear response in the concentration range from 0.75 to 7.5 μmol/L and a limit of detection of 0.175 μmol/L using Electrochemical Impedance Spectroscopy (EIS) (R2 = 0.989) and 0.513 μmol/L using Cyclic Voltammetry (CV) (R² = 0.953) for synthetic melatonin. Furthermore, the sensors exhibited a good stability and reproducibility (3.45% and 2.87% for EIS and CV, respectively, n = 3), maintaining adequate response even after 30 days of assembly. On biologically-present melatonin-containing samples the device displayed a similar performance when compared to ELISA technique (deviation of 13.31%). We expect that the developed device contributes significantly to the medical area allowing precise and complete diagnosis of the diseases related to abnormal levels of melatonin.

The adverse effect of gold-based nanoparticles is still an open question since it depends on several factors as shape, surface charge or route of administration. In this study, we investigated the influence of shape and human serum albumin (HSA) coating on the adverse effects of spherical (AuNP) and nanorods (AuNR) gold-based particles. F C3H (fibroblast) and HTC (hepatocellular carcinoma) cell lines both from liver were exposed to 25, 75 and 125 μg mL−1, which correspond to 109 NP mL−1. For in vivo studies, Wistar rats received these materials by oral administration in doses of 10 μg kg−1 or 40 μg kg−1. Systemic toxicity was verified after 24 h and 48 h by morphological analysis, blood parameters and myeloperoxidase enzyme activity. Our results revealed that HSA corona does not influence totally the pathway of interactions between AuNP and AuNR. In vitro results evidenced that AuNP can decrease in at least 50% viability of F C3H and cell adhesion of HTC, but corona significantly overcomes these effects. No differences between shape or corona were observed in function of cell lines. In vivo studies showed that 40 μg kg−1 of AuNP-HSA caused an enhancement of the myeloperoxidase response indicating inflammatory processes. An increase from 40% to 80% on alkaline phosphatase levels were found for all groups. Our findings suggested that gold-based particles coated or not with HSA do not cause expressive adverse effects on in vitro or in vivo systems, and their oral administration cannot cause a systemic effect in the experimental conditions used here.

Sickle cell anemia (SCA) is a common recessive genetic condition in which patients produce an abnormal form of hemoglobin. The disease is common mainly among African individuals and in parts of the continent up to 40 % of the population presents its genetic trait. Currently, disease diagnosis and trait determination are performed using polymerase chain reaction, liquid chromatography and electrophoresis. Although these methods present high sensitivity and are well established, they are costly and require specialized equipment to be performed. We developed an electrochemical genosensor for simple and low cost SCA trait determination. The device was based on the immobilization of single DNA strands containing the disease related mutation on gold platforms using the self-assembled monolayers technique. The determination of SCA trait was then performed using electrochemical impedance spectroscopy. The genosensor displayed a wide linear range (0.01 to 7.5 μmol L−1, R2=0.979), with a detection limit of 7.0 nmol L−1. Furthermore, the device was able to distinguish between DNA sequences containing or not the mutation (target and non-target sequences) with precision and great reproducibility (10.4 %, n=3). It is expected that such sensor increases the number of SCA trait determination, promoting early diagnosis and genetically counseling.

Understanding the interactions between biomolecules and nanomaterials is of great importance for many areas of nanomedicine and bioapplications. Although studies in this area have been performed, the interactions between cell membranes and nanoparticles are not fully understood. Here, we investigate the interactions that occur between the Langmuir monolayers of dipalmitoylphosphatidyl glycerol (DPPG) and dipalmitoylphosphatidyl choline (DPPC) with gold nanorods (NR)-with three aspect ratios-and gold nanoparticles. Our results showed that the aspect ratio of the NRs influenced the interactions with both monolayers, which suggest that the physical morphology and electrostatic forces govern the interactions in the DPPG-NR system, whereas the van der Waals interactions are predominant in the DPPC-NR systems. Size influences the expansion isotherms in both systems, but the lipid tails remain conformationally ordered upon expansion, which suggests phase separation between the lipids and nanomaterials at the interface. The coexistence of lipid and NP regions affects the elasticity of the monolayer. When there is coexistence between two phases, the elasticity does not reflect the lipid packaging state but depends on the elasticity of the NP islands. Therefore, the results corroborate that nanomaterials influence the packing and the phase behavior of the mimetic cell membranes. For this reason, developing a methodology to understand the membrane-nanomaterial interactions is of great importance.

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices. Copyright: © 2016 Beal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Regardless the controversies surrounding genetically modified organisms (GMO), their cultivation isconstantly increasing and in according to the EU legislation, labeling is mandatory for products con-taining EU-authorized-GMO higher than 0.9%. Thereby, new analytical strategies for rapid and effectivedetection of GMO on foodstuffs are required. In this work, an electrochemical immunosensor foreffective determination of Cry1Ab protein from MON810 transgenic maize (EU-authorized-GMO) isdescribed. The immunosensor was developed onto indium tin oxide (ITO) electrodes modified by3-aminopropyltrimethoxysilane (APTES) monolayer to covalently immobilize Anti-Cry1Ab polyclonalantibodies. The protein interaction with the polyclonal antibody (PAb) recognition platform was directlymonitored and measured by cyclic voltammetry and electrochemical impedance spectroscopy usingcommercially Cry1Ab protein. After the analytical features optimization a linear response from 1 to10 ng mL−1, a limit of detection (LOD) of 0.37 ng mL−1and a limit of quantification (LOQ) of 1.23 ng mL−1– which provided accurate results (RSD < 7.5%) – were achieved. The immunosensor allowed a simple andfast detection of Cry1Ab protein extracted from maize seeds with different GM maize mass percentages(0%, 0.5%, 1%, 2.5% and 5%). To crosscheck the detection of Cry1Ab protein, an enzyme-linked immunosor-bent assay (ELISA) was used. The results indicate that the immunosensor is suitable for the transgenicprotein Cry1Ab detection in GM maize representing a successfully tool to verify the compliance of theEU regulations.

The development of new systems for diagnostic and therapeutic applications is a topic of intense and growing interest. The unique optical and electronic properties exhibited by gold-based nanomaterials have proved to be valuable for a wide range of biomedical applications. Recently, great attention has been given to rod-shaped gold nanomaterials, especially because of their electronic absorption band in the near infrared region, where maximum radiation penetration through tissue occurs. This feature has allowed the use of gold nanorods for in vivo imaging, with different techniques, and photothermal therapy. Furthermore, gold nanorods can be functionalized with a wide variety of biomolecules for cancer cell targeting. Moreover, their versatility and unique properties have generated much enthusiasm in medicine. The present review aims to show the recent advances in the synthesis and applications of gold nanorods in medical areas.

The viability of a fiber optic reflection-based Localized Surface Plasmon Resonance (LSPR) sensor using layer-by-layer technique composed by PAMAM-AuNP with and without AuNP-citrate was investigated. The PAMAM-AuNPs and PAMAM-AuNPs/AuNP-citrate layers were deposited on the endface of an optical fiber and the reflected signal was acquired. Deposition time and number of layers were optimized viewing LSPR sensing applications. Results with and without AuNP-citrate were compared. The sensor is being characterized as a refractive index sensor.

Sickle cell anemia (SCA) is a common recessive genetic condition in which patients produce hemoglobin S (HbS), an abnormal form of the protein, instead of hemoglobin A, the regular form. SCA is caused by the substitution of a single nitrogenous base adenine for thymine (GAG-> GTG), encoding valine instead of glutamine.1 Deoxygenation of HbS leads to its polymerization, damaging the cell membrane and reducing the lifetime of erythrocytes dramatically – which, in turn, leads to anemia. In some regions of Africa, up to 40% of the population possess its genetic trait, being considered a public health serious problem2. As the disease is possibly deadly, presents very low cure chances and has limited treatment, carriers determination is a key feature to assist in reproducibility decisions. Currently, trait determination is mainly performed by liquid chromatography, electrophoresis and polymerase chain reaction (PCR)3,4. These techniques are not widely used due to its complexity, lack of portability, high price and specialized personal and equipment demand. In this study we have developed an electrochemical genosensor for simple, low cost and rapid SCA carriers determination. Single DNA strands corresponding to the sequence of a SCA carrier were immobilized in gold platforms using the self-assembled-monolayers technique. The determination of SCA trait is then performed by analyzing the film’s charge transfer resistance using Electrochemical Impedance Spectroscopy. The genosensor was able to distinguish between healthy and unhealthy subjects with great precision besides displaying a wide linear range (0.01 to 7.5 µmol/L, R2 = 0.927). Furthermore, the device presented a detection limit of 7.0 nmol/L. We expect that such devices increases the number of SCA carriers determination, promoting early diagnosis and genetical counseling. References: 1 Barany, F. Genetic-Disease Detection and DNA Amplification Using Cloned Thermostable Ligase. Proceedings of the National Academy of Sciences of the United States of America 88, 189-193, doi:10.1073/pnas.88.1.189 (1991). 2 Diallo, D. & Tchernia, G. Sickle cell disease in Africa. Current Opinion in Hematology 9, 111-116, doi:10.1097/00062752-200203000-00005 (2002). 3 Ayatollahi, M. & Haghshenas, M. Application of the polymerase chain reaction to the diagnosis of sickle cell disease in Iran. Archive of Iranian Medicine 7, 84-88 (2004). 4 Bender, M. A. & Seibel, G. D. Sickle Cell Disease. (University of Washington, 2003).

Sistemas de liberação em nanoescala, capazes de identificar e responder ativamente e com precisão a estímulos externos ou internos ao microambiente celular visando uma cinética de liberação sob demanda, são de grande interesse para a liberação de compostos terapêuticos. No entanto, para o desenvolvimento desses sistemas, é necessária uma profunda compreensão da interação entre nanomaterial e células. No capítulo I desta tese, descrevemos como nanogéis (NGs) de acrilamida no seu estado intumescido e com diferentes densidades de reticulação, (e consequentemente, diferente rigidez) são internalizados por uma monocamada polarizada de células endoteliais microvasculares cerebrais humanas (hCMEC / D3), e como isso se correlaciona com sua capacidade de atravessar a barreira hematoencefálica (BBB). Os NGs mais rígidos mostraram uma maior captação pela camada celular polarizada, enquanto os NGs mais elásticos exibiram maior capacidade em atravessar a BBB sem comprometer a permeabilidade da monocamada, sendo que 8,2, 7,5 e 5,2% dos NGs atingiram o compartimento basolateral após 16 horas de incubação a 37 ° C em atmosfera úmida com 5% de CO2. Também observamos que a taxa de transporte dos NGs é elevada nas duas horas iniciais de interação. Os mesmos NGs descritos no Capítulo I foram empregados em um segundo estudo relatado no Capítulo II, para avaliar a resposta da rigidez em monocultura 2D e na co-cultura direta de células de glioma, C6, e macrófagos, J774, em relação à internalização e à citotoxicidade dos NGs. Os resultados apontam uma internalização favorecida pelos NGs mais rígidos, e um efeito tóxico mais elevado do NG mais elástico. O capítulo III aborda o desenvolvimento e a avaliação in vitro de nanocarreadores (NCs) revestidos por membrana de células de câncer de mama (MCF-7), compostos de poli (D, ácido L-láctico-co-glicólico) (PLGA) e contendo paclitaxel (PTX) como fármaco modelo. O carreamento do fármaco em PLGA-PTX NCs foi de aproximadamente 4 wt% (98 ± 1% da eficiência do encapsulamento) e apresentou redução significativa da viabilidade celular contra células de câncer de mama quando comparado aos NCs não revestidos (interação homotípica) e ainda, exibiu um aumento da associação de NCs revestidos por membrana com outros tipos de células epiteliais. O Capítulo IV descreve o projeto e desenvolvimento de um sistema de liberação sob demanda induzido pela luz, empregando motores moleculares hidrofóbicos (MM) para desestabilizar a bicamada lipídica de lipossomos compostos por lipídios insaturados. Demonstramos que a combinação dos lipossomas com o MM resultou em um sistema de liberação sensível à luz controlável. Nanoscale delivery systems capable of actively identify and respond precisely to external stimuli or internal cues in the cellular microenvironment aiming on demand kinetics release are of great interest to the delivery of therapeutic compounds. However, to be able to create these systems a deep understanding of nanomaterial-cell interaction is necessary. In the Chapter I of this thesis, we describe how swollen acrylamide nanogels (NGs) with different cross-linking densities and, consequently, different stiffnesses are internalized by a polarized monolayer of human cerebral microvascular endothelial cells (hCMEC/D3) and how does it correlate with their ability to cross the blood-brain barrier (BBB) using a filter-free in vitro BBB model. The harder NGs showed a higher uptake by the polarized cell layer while the softer NGs exhibited an enhanced capacity of crossing the BBB without affecting the monolayer permeability with 8.2, 7.5 and 5.2 % of NGs reaching the basolateral compartment after 16 hours incubation at 37 °C in humidified atmosphere with 5% CO2. We also observed that NGs transport rate is elevated in the first 2 hours of interaction. The same NGs described in Chapter I were employed in a second study reported in Chapter II to assess the effect of their varied stiffness in 2D monoculture and direct coculture of C6 glioma cells and J774 macrophages regarding NGs internalization and cytotoxicity. The results showed a preferred internalization of harder NGs but a higher toxic effect from the softer NG, NG1.5. Chapter III addresses the development and in vitro evaluation of MCF-7 breast cancer cell membrane-coated nanocarriers (NCs) composed of poly (D, L-lactic-co-glycolic acid) (PLGA) and containing paclitaxel as a drug model. The PLGA-PTX NCs drug loading is roughly 4 wt% (98 ± 1% of encapsulation efficiency) and presented significant reduction of cell viability against breast cancer cells when compared to the non-coated NCs (homotypic interaction), and also reveals the increased membrane-coated NCs association with other epithelial cell types. To conclude, Chapter IV describes the design and development of a light-induced on demand release system employing hydrophobic molecular motors (MM) to destabilize the lipid bilayer of liposomes composed of unsaturated lipid. We demonstrate that the combination of the liposomes with the MM resulted in a controllable light-sensitive release system.

O câncer de mama é a segunda maior causa de morte em mulheres em todo o mundo. Nas últimas décadas, a terapia fotodinâmica (TFD) tem sido investigada em estudos clínicos como uma alternativa minimamente invasiva para recidivas em pele na parede torácica após mastectomia. Para entender os efeitos da TFD em tumores sólidos, estudos in vitro em modelos que mimetizem a morfologia e ambiente natural de ocorrência do tumor são essenciais. Nesse contexto, existem modelos de cultura tridimensional (3D), como o método de levitação magnética (MLM) que permite a formação de esferoides da ordem de milímetros em poucos dias. Os objetivos deste trabalho foram caracterizar o crescimento dos tumores no MLM com MCF-7, uma linhagem de células de câncer de mama, investigar citotoxicidade e distribuição dos fotossensibilizadores (FS) nesse modelo, avaliar os efeitos da TFD nas culturas 3D milimétricas in vitro e em modelo de membrana corioalantóica (CAM). A protoporfirina IX (PpIX) e um derivado de clorina e6 (PDZ) foram utilizados nas investigações de citotoxicidade e distribuição. Os protocolos de TFD foram realizados com PDZ ativado por baixas fluências de luz (1, 3 e 4 J/cm2), para obtenção de tumor remanescente, entregues por uma fonte de luz emitindo em 660 nm. Nossos resultados mostraram que com o MLM foi possível gerar tumores do tamanho de interesse (2,39 ± 0,04 mm de diâmetro aparente médio) e foi observado que a PpIX não foi distribuída por todo tumor nos parâmetros analisados. Já o PDZ se distribuiu homogeneamente por todo tumor na concentração de 100 μg/mL e com 50 μg/mL foi possível reproduzir uma distribuição heterogênea do FS como ocorre in vivo. Protocolos de TFD com baixa fluência de luz foram padronizados para gerar baixa taxa de morte e destruição parcial dos tumores. Nos protocolos de uma sessão de 3 J/cm2 e 3 sessões de 1 J/cm2, a microspectroscopia Raman identificou sinais de apoptose a curto prazo e dano na matriz extracelular a longo prazo. Nossos resultados demonstraram possíveis efeitos da TFD em tumores sólidos quando há presença de tumor remanescente. Acreditamos que esses resultados podem auxiliar futuros estudos referentes à resistência de tumores à TFD, aumento de agressividade e metástase. Os testes em CAM foram inconclusivos, mas sugerem a possibilidade de interação do tumor com a membrana. Breast cancer is the second leading cause of death in women around the world. In the last decades, Photodynamic Therapy (PDT) has been investigated in clinical studies as a minimally invasive alternative for chest wall recurrence after mastectomy. To understand the effects of PDT on solid tumors, in vitro studies on models that mimic the morphology and natural environment of the tumor are essential. In this context, there are three-dimensional (3D) culture models, such as the magnetic levitation method (MLM) that allows formation of milimetric tumors in few days. The purpose of this work were to characterize the growth of MLM tumors with MCF-7, a breast cancer cell line, to investigate cytotoxicity and distribution of the photosensitizer (PS) in the model, to evaluate the effects of PDT on millimetric 3D cultures in vitro and in a model of chorioallantoic membrane (CAM). Protoporphyrin IX (PpIX) and a chlorin e6 derivative (PDZ) were used for the cytotoxicity assay and distribution investigations. The PDT protocols were performed with PDZ activated by low fluences of light (1, 3 e 4 J/cm2), aiming to obtain a remaining tumor, delivered by a light source emitting at 660 nm. Our results showed that with the MLM it was possible to generate tumors of interest size (2.39 ± 0.04 mm of mean diameter). PDZ was homogeneously distributed throughout the tumor at a concentration of 100 μg/mL, and at 50 μg/mL it was possible to reproduce a heterogeneous distribution of the PS as seen in vivo. Low-fluence PDT protocols were standardized to generate death rate and partial destruction of tumors. After the protocols of one session of 3 J/cm2 and 3 sessions of 1 J/cm2, Raman microspectroscopy identified signs of apoptosis in short-term and extracellular matrix damage in long-term. Our results demonstrated some possible effects of PDT on solid tumors when there is presence of tumor remaining. We believe that these results may support future studies regarding tumor resistance to PDT, increase of aggressiveness and metastasis. The CAM tests were inconclusive, but there is a possibility of tumor-to-membrane interaction.

Em 1978, o trabalho realizado por Stephenson e Zamecnik demonstrou a capacidade de um oligonucleotídeo de impedir a expressão de uma proteína específica. Atualmente, duas tecnologias são mais utilizadas para este propósito: os oligodeoxiribonucleotídeos antisense e o RNA de interferência (siRNA), que se aproveitam da capacidade de anelação entre as fitas complementares. A maior diferença entre as duas técnicas é a maquinaria proteica recrutada, isso é, o complexo RISC atua no funcionamento do siRNA, e a protease RNase H atua na clivagem da fita de RNA quando hibridizada com DNA. Apesar da grande aplicabilidade destas tecnologias, tanto para doenças metabólicas quanto para canceres, o veículo de entrega e proteção dessas sequências é de fundamental importância, visto que a aplicação desses oligonucleotídeos livres está sujeita à rápida degradação e ineficiência. A modificação das bases é uma das estratégias para conferir maior estabilidade às sequências, porém estas tem sido relacionadas a um aumento da toxicidade. Nessa dissertação, a quitosana, um polissacarídeo catiônico é utilizado para síntese de nanopartículas e encapsulamento dos oligodeoxiribonucleotídeos antisense (ASO). Para isso, foram realizadas modificações na quitosana comercial como despolimerização, trimetilação ou conjugação com PEG, seguida da síntese das nanopartículas com a adição de tripolifosfato de sódio (TPP) pelo método de gelatinização ionotrópica. A estabilidade das nanopartículas foi medida em função do tempo, da variação de temperatura e da diferença de pH. Além disso, a toxicidade dessas nanopartículas foi analisada através da viabilidade celular em diferentes linhagens, NB-4, HepaRG, HTC e BHK-570. A expressão da proteína verde fluorescente (GFP) na célula NB-4 foi utilizada para avaliar a entrega do ASO desenhado, sendo sua fluorescência monitorada por microscopia confocal. Os resultados demonstram que as nanopartículas se mantiveram estáveis durante o período de tempo analisado, assim como com a temperatura variando de 22 a 45°C e em pH ácido. Cada linhagem celular respondeu de forma diferente ao tratamento com as nanopartículas sem ASO, sendo a linhagem saudável BHK-570 com a maior resistência. Ademais, todas as células apresentaram viabilidade reduzida quando tratadas com concentrações na ordem de 1011 nanopartículas/mL a base de quitosana trimetilada. A fluorescência das células NB-4 quando tratada com as nanopartículas com ASO diminuiu consideravelmente nas 18 primeiras horas, seguida de um aumento após 42 horas. Dessa forma, pode-se concluir que as nanopartículas de quitosana propostas nessa dissertação apresentaram uma excelente alternativa para a entrega de material genético, principalmente para o trato gastro-intestinal, devido à sua estabilidade em pH ácido. The property of an oligonucleotide to interfere in the expression of a protein was observed in 1978 by Stephenson and Zamecnik. To perform such interference, there are today, two main techniques being explored: antisense oligodeoxyribonucleotides and interference RNA. In both cases, the particularity of their chemical structure is taken into account as soon as they can bind in a complementary manner to the messenger RNA and inhibit its translation. The great difference between these techniques is related to the proteases involved in the process, while for interference RNA the RISC machinery acts, for antisense oligodeoxyribonucleotides RNase H cleaves the RNA in the duplex DNA-RNA. Although these tools to edit the translation process are relevant to the treatment and even cure of metabolic disorders and cancers, it is still not effective when employed without a coating to protect the sequences before it reaches the destiny in vivo. Efforts have been made in developing modified bases to be more stable, but they show some toxicity. In this dissertation, chitosan, a natural cationic polyssacharide, is used to produce nanoparticles to protect the antisense oligodeoxyribonucleotide (ASO). For this reason, the commercial chitosan was modified, depolymerized, trimetilated or PEGlated and the nanoparticles were synthesized with sodium tripolyphosphate (TPP) by ionotropic gelation method. The stability along time, in different pHs and temperatures was assessed. The toxicity of nanoparticles without ASO was quantified by MTT tests in NB-4, HepaRG, HTC and BHK-570 cell lines. A green fluorescent protein (GFP) expressed by NB-4 cells was the target to evaluate the delivery efficiency of the ASO, and its fluorescence was measured by confocal microscopy. Results showed that nanoparticles were stable over time as well as in temperatures ranging from 22 to 45°C and in acidic pH. Each cell line responded in a different manner to the treatment, with the health cell BHK-570 showing higher resistance. Furthermore, all of them presented lower viability when treated with trimetilated chitosan nanoparticles in the highest concentrations (ca 1011 nanoparticles/mL). NB-4 cells presented a decrease in fluorescence in 18 hours of treatment followed by an increase after 42 hours. We conclude that chitosan nanoparticles are a good alternative to the delivery of genetic material even more in the gastro intestinal tract due to its great stability in acid pH values.

Nanopartículas de ouro têm mostrado enorme potencial de aplicação em modalidades diagnósticas e terapêuticas fotoativadas. Em especial, nanoestruturas de ouro anisotrópicas ramificadas apresentam excelente desempenho atuando tanto como contrastes de imagens fotoacústicas, quanto como agentes ativos para terapias fototérmicas de câncer. Apesar dos avanços nas suas rotas de síntese, o desenvolvimento dessas nanoestruturas de forma simples e reprodutível ainda é desafiador. O presente trabalho visou o desenvolvimento de nanopartículas de ouro anisotrópicas ramificadas, ou nanoflores, que sejam fotoativas no infravermelho-próximo para a terapia e diagnóstico de câncer. Em particular, buscou-se o desenvolvimento de uma síntese simples para sua obtenção, assim como a verificação de sua atuação como agente de contraste fotoacústico e como agente ativo para hipertermia de tumores. Para tanto, desenvolveu-se uma síntese in situ que permitiu a obtenção de nanoflores monodispersas com tamanho e propriedades ópticas controláveis. Através da variação de aspectos da síntese, como a temperatura e a concentração de ouro, foi possível sintonizar a atividade óptica das partículas entre 590 e 960 nm. Sua formação foi confirmada por microscopia eletrônica de varredura, espalhamento de luz dinâmico e espectroscopia UV-visível. As partículas apresentaram boa estabilidade de suas características físico-químicas por dois meses e meio. Ainda, as nanoflores se mostraram estáveis, também, quando suspensas em meio de cultura, sob irradiação de lasers, e quando mantidas a temperatura corpórea por longos intervalos. Sua resposta fotoacústica foi caracterizada, apresentando sinais significativos e permitindo a obtenção de imagens claras de sua localização, mesmo em baixas concentrações. Testes realizados em cultura de células mostraram que as nanoflores foram eficazes na hipertermia de uma linhagem de hepatocarcinoma de rato (HTC), ao mesmo tempo que não apresentaram sinais de toxicidade a uma linhagem de fibroblastos de camundongos (FC3H). Esses resultados revelam uma possibilidade simples de fabricação de nanoestruturas de ouro anisotrópicas ramificadas, que podem servir como uma plataforma promissora para o diagnóstico e terapia do câncer. Gold nanoparticles have shown enormous potential of application in photodiagnostic and in phototherapeutic procedures. Notably, branched anisotropic gold nanostructures present distinguished performance acting as contrast agents of photoacoustic images and as active agents for photothermal therapies for cancer. Despite advances in their synthesis routes, the growth of these nanostructures in a simple and reproducible way is still challenging. The present study was aimed at developing branched anisotropic gold nanoparticles, coined nanoflowers, that are photoactive in the near-infrared for therapy and diagnosis of cancer. In particular, we sought to develop a simple synthesis route, as well as to verify its application for both, as photoacoustic contrast agents and as active agents for tumor hyperthermia. An in situ synthesis was developed which allowed the development of monodisperse nanoflowers with controllable size and optical properties. Through variations of certain aspects of this procedure, such as temperature and gold ions concentration, it was possible to tune the optical activity of the particles between 590 and 960 nm. The nanostructure morphology was confirmed by scanning electron microscopy, dynamic light scattering and UV-visible spectroscopy. The particles exhibited consistent physicochemical characteristics and good stability for two and a half months. Furthermore, the nanoflowers were also stable when suspended in cell culture medium, under laser irradiation and when maintained at body temperature for long intervals. Its photoacoustic response was characterized, presenting significant responses and generating clear images of its location, even at low concentrations. In vitro tests revealed that these nanoflowers were effective therapeutic agents for photothermal therapy of a rat hepatocarcinoma (HTC) lineage, while showing no signs of toxicity to mouse fibroblast (FC3H) cell line. These results reveal a simple procedure of synthesizing branched anisotropic gold nanostructures, which can serve as a promising platform for cancer diagnosis and therapy.