Estudo da encapsulação de fármacos antineoplásicos em sistemas nanoestruturados: Caracterização estrutural e efeito sinérgico de fármacos / Study of the encapsulation of antineoplastic drugs in nanostructured systems: Structured characterization and synergistic effect of drugs

Dentro da área da nanotecnologia, o sistema drug delivery vem sendo amplamente utilizado, cujo objetivo é proporcionar uma maior eficácia dos ativos farmacêuticos, podendo envolver desde uma distribuição mais seletiva dentro do organismo até a taxa que as moléculas serão liberadas e/ou a atenuação dos efeitos adversos provocados. Para isso, os ativos são encapsulados em nanoestruturas, podendo estas serem de natureza sintética ou natural. Dentre os nanocarreadores promissores encontram-se os cubossomos, que são nanoestruturas complexas capazes de encapsular ativos tanto hidrofílicos quanto hidrofóbicos. O objetivo deste projeto foi estudar a encapsulação de fármacos antineoplásicos em sistemas drug delivery contra linhagens celulares, investigando também as alterações estruturais sofridas pelos cubossomos e os efeitos sinérgicos dos fármacos, sendo eles: a doxorrubicina, a cisplatina, a vemurafenibe e a curcumina. As metodologias empregadas para elucidar o efeito das combinações dos fármacos, a estruturação da nanopartícula e sua citotoxicidade foram: os estudos de viabilidade celular pós-exposição, espalhamento dinâmico de luz, potencial zeta, análise de rastreamento de nanopartículas, espalhamento de raios-x a baixos ângulos, criomicroscopia eletrônica de transmissão, eficiência de encapsulação e ensaio de liberação. Inicialmente os fármacos foram testados isoladamente e em duplas, sendo utilizadas cinco linhagens celulares, afim de se promover um delineamento aos ensaios futuros. A partir destes resultados, foi-se optado por manter duas linhagens celulares, a HeLa, como representante de tecidos tumorais, e a HaCat, modelo de tecido saudável, devido a menor resistência apresentada por elas. Em relação as combinações entre as drogas, pode-se observar que todas as duplas formadas apresentaram resultados sinérgicos na linhagem tumoral, sendo mantida para os testes seguintes a combinação curcumina e vemurafenibe. Os cubossomos foram sintetizados eficientemente, sendo produzidos na ausência de fármacos bem como contendo curcumina e vemurafenibe. As nanopartículas apresentaram uma variação de diâmetro entre 189 ± 3 nm e 224 ± 2 nm, sendo o PDI entre 0,08 e 0,25. A conformação do cubossomo foi confirmada através da criomicroscopia eletrônica de transmissão e pelo espalhamento de raios-x a baixos ângulos, onde foi determinada uma estruturação característica de Pn3m. Para a eficiência de encapsulação os valores variaram entre 79% de encapsulação para a curcumina e 72% para a vemurafenibe, quando utilizadas isoladamente. No caso da encapsulação em dupla, os valores se converteram para 63% e 53% para a curcumina e vemurafenibe, respectivamente. A liberação das drogas do interior da nanopartícula oscilou entre 1500, 480 e 420 minutos para os cubossomos de curcumina, vemurafenibe e curcumina + vemunafenibe, respectivamente. Os testes de citotoxicidade demonstraram que as concentrações de 0,01 e 0,03 mg/mL foram capazes de promover uma viabilidade acima de 70%, porém, utilizando estas proporções não foi possível observar resultados significativos. Por fim, o sistema se mostrou estável e homogêneo, sendo capaz de promover a encapsulação dos fármacos tanto singularmente quanto em dupla e, apesar da quantidade de fármacos não ter sido suficiente para ocasionar alterações ao sistema celular, a execução deste trabalho abre portas para que novos estudos sejam realizados, podendo-se testar diferentes ativos bem como alterando a composição da nanopartícula afim de se reduzir a citotoxicidade

Within the area of nanotechnology, the drug delivery system has been widely used, whose objective is to provide greater effectiveness of pharmaceutical active ingredients, which may range from a more selective distribution within the organism to the rate at which the molecules will be released and/or the attenuation of adverse effects caused. To achieve this, the active ingredients are encapsulated in nanostructures, which may be synthetic or natural in nature. Among the promising nanocarriers are cubosomes, which are complex nanostructures capable of encapsulating both hydrophilic and hydrophobic active ingredients. The objective of this project was to study the encapsulation of antineoplastic drugs in drug delivery systems against cell lines, also investigating the structural changes undergone by the cubosomes and the synergistic effects ofthe drugs, namely: doxorubicin, cisplatin, vemurafenib and curcumin. The methodologies used to elucidate the effect of drug combinations, the structuring of the nanoparticle and its cytotoxicity were: post-exposure cell viability studies, dynamic light scattering, zeta potential, nanoparticle tracking analysis, small angle x-rays scattering, transmission electron cryomicroscopy, encapsulation efficiency and release assay. Initially, the drugs were tested alone and in pairs, using five cell lines, in order to promote a design for future trials. Based on these results, it was decided to maintain two cell lines, HeLa, as a representative oftumor tissues, and HaCat, a model ofhealthy tissue, due to their lower resistance. Regarding the combinations between the drugs, it can be observed that all the pairs formed presented synergistic results in the tumor lineage, with the combination of curcumin and vemurafenib being maintained for the following tests. Cubosomes were efficiently synthesized, being produced in the absence of drugs as well as containing curcumin and vemurafenib. The nanoparticles varied in diameter between 189 ± 3 nm and 224 ± 2 nm, with the PDI being between 0.08 and 0.25. The conformation ofthe cubosome was confirmed through transmission electron cryomicroscopy and small angle x-rays scattering, where a characteristic structure of Pn3m was determined. For encapsulation efficiency, values varied between 79% encapsulation for curcumin and 72% for vemurafenib, when used alone. ln the case of double encapsulation, the values converted to 63% and 53% for curcumin and vemurafenib, respectively. The release of drugs from the interior of the nanoparticle ranged between 1500, 480 and 420 minutes for the curcumin, vemurafenib and cubosomes with curcumin + vemunafenib, respectively. Cytotoxicity tests demonstrated that concentrations of 0.01 and 0.03 mg/mL were capable of promoting viability above 70%, however, using these proportions it was not possible to observe significant results. Finally, the system proved to be stable and homogeneous, being able to promote the encapsulation of drugs both singly and in pairs and, although the quantity of drugs was not enough to cause changes to the cellular system, the execution of this work opens doors for new studies are carried out, with the possibility oftesting different active ingredients as well as changing the composition of the nanoparticle in order to reduce cytotoxicity

Caracterização de cubossomos não-iônicos em presença de proteínas modelo: uma abordagem estrutural e funcional / Characterization of non-ionic cubosomes in the presence of model pro­teins: a structural and functional approach

Uma área de pesquisa que vem ganhando muita atenção nos últimos anos é a nanome­dicina, com especial atenção para os sistemas com entrega controlada de fármacos, ou drug delivery. Dentre as diversas nanopartículas utilizadas para este fim, destacam-se os sistemas formados por lipídeos e polímeros, como por exemplo os lipossomos e os cubossomos. Neste trabalho, é estudada a influência estrutural da lisozima e da curcumina, proteínas modelo. A lisozima é uma enzima antimicrobiana produzida por animais e que faz parte do sistema imunológico. Ela é uma hidrolase glicosídica que catalisa a hidrólise dos componentes da parede celular de bactérias gram-positivas. Esta hidrólise, por sua vez, compromete a integridade das paredes celulares, causando a lise (e como consequência a morte) das bactérias. Curcumina é um composto cristalino de cor amarelada brilhante, encontrada no caule da Curcuma longa (ou açafrão), que tem sido utilizada como corante ou até mesmo como aditivo alimentar. Este composto tem sido uma grande aposta no tratamento de doenças crônicas como inflamação, artrite, síndrome metabólica, doença hepática, obesidade, doenças neurodegenerativas e principalmente canceres, sendo também utilizada em estudos como potencial agente antibacteriano. O principal objetivo deste trabalho é construir sistemas nanoestruturados com potencial de atuarem como sistemas antimicrobianos, com a liberação controlada de ambos dos fármacos. Estes sistemas são compostos por cubossomos de fitantriol (PHY) em ausência e presença da lisozima, da curcumina e de suas combinações, a fim de analisar ação antimicrobiana conjunta da lisozima e da curcumina. As técnicas biofísicas utilizadas para caracterizar essas partículas são SAXS (espalhamento de raios-X em baixos ângulos), DLS (espalhamento dinâmico de luz), Cryo-TEM (criomicroscopia eletrônica de transmissão) e NTA (análise de rastreamento de nanopartículas). Foi possível verificar que as formulações lipídicas são eficazes na formação de estruturas cúbicas com estabilidade desejável. As nanopartículas cúbicas demonstraram alta capacidade de encapsulação da lisozima e da curcumina. A cinética de liberação desses medicamentos mostrou-se promissora, sugerindo que a encapsulação dos fármacos é eficaz, bem como a liberação controlada e direcionada. Duas linhagens de bactérias foram estudadas, sendo que a E. coli, não sofreu nenhum dano citotóxico, enquanto a Bacillus subtilis sim. Tal resultado indica o potencial antimicrobiano do sistema para alguns tipos de bactérias

An area of research that has gained significant attention in recent years is nanomedicine, with a particular focus on drug delivery systems. Among the various nanoparticles used for this purpose, lipid and polymer-based systems, such as liposomes and cubosomes stand out. This study investigate the structural influence of encapsulating lysozyme and curcumin, model compounds. Lysozyme is an antimicrobial enzyme produced by animals and is part of the immune system. It is a glycosidic hydrolase that catalyzes the hydrolysis of components in the cell walls of gram-positive bacteria. This hydrolysis compromises the integrity of cell walls, leading to the lysis (and consequently the death) of bacteria. Curcumin is a bright yellow crystalline compound found in the stem of Curcuma longa (or turmeric), commonly used as a dye or even as a food additive. It has been a significant focus in the treatment of chronic diseases such as inflammation, arthritis, metabolic syndrome, liver disease, obesity, neurodegenerative diseases, and especially cancers. It is also studied as a potential antibacterial agent. The main objective of this study is to construct nanostructured systems with the potential to act as antimicrobial agents, with controlled release of both drugs. These systems consist of phytantriol (PHY) cubosomes in the absence and presence of lysozyme, curcumin, and their combinations to analyze the joint antimicrobial action of lysozyme and curcumin. Biophysical techniques used for characterization include Small-Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Cryo-Transmission Electron Microscopy (Cryo-TEM), and Nanoparticle Tracking Analysis (NTA). It was observed that lipid formulations are effective in forming cubic structures with desirable stability. Cubic nanoparticles have demonstrated a high encapsulation capacity for lysozyme and curcumin. The release kinetics of these drugs have shown promise, suggesting that drug encapsulation is effective, as well as their controlled and targeted release. Two bacterial strains were studied, with E. coli showing no cytotoxic damage, while Bacillus subtilis did. This result indicates the antimicrobial potential of the system against types of bacteria

Cryo-EM structures for the Mycobacterium tuberculosis iron-loaded siderophore transporter IrtAB

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.

Knockout of ribosomal genes bS22 and bL37 increases the sensitivity of mycobacteria to antibiotics / 生物工程学报

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc2155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.

Humberto Fernández-Morán (1924-1999) a pioneer in electron ultra-cryomicroscopy / Humberto Fernández-Morán (1924-1999) pionero en ultracriomicroscopía electrónica

SUMMARY: Humberto Fernández-Morán (1924-1999) a Venezuelan physician and biophysicist research, who developed the diamond knife. Furthermore he focused on improving the mechanical performance, accuracy and reliability of mocrotomes and ultramicrotomes which significantly advanced the development of electromagnetic lenses for electron microscopy based on superconducting technology. Promoter and founded of the Venezuelan Institute for Neurological and Brain Studies. He was a pioneer in electron ultra-cryomicroscopy field. Fernández-Morán taught and researched in University of Stockholm, Massachusetts Institute of Technology, Harvard University, Massachussetts General Hospital and the University of Chicago. He worked with NASA for the Apollo project in the field of physic-chemical analysis of lunar rocks.

RESUMEN: Humberto Fernández-Morán (1924-1999) médico venezolano e investigador biofísico, quien desarrolló el cuchillo de diamante. Además, se centró en mejorar el rendimiento mecánico, la precisión y la fiabilidad de los micrótomos y ultramicrótomos, lo que avanzó significativamente en el desarrollo de lentes electromagnéticos para microscopía electrónica basados en tecnología superconductora. Promotor y fundador del Instituto Venezolano de Estudios Neurológicos y Cerebrales. Fue pionero en el campo de la ultracriomicroscopía electrónica. Fernández-Morán enseñó e investigó en la Universidad de Estocolmo, el Instituto Tecnológico de Massachusetts, la Universidad de Harvard, el Hospital General de Massachussets y la Universidad de Chicago. Trabajó con la NASA para el proyecto Apollo en el campo del análisis físico-químico de rocas lunares.

Progress in filters for denoising cryo-electron microscopy images / 北京大学学报(医学版)

Cryo-electron microscopy (cryo-EM) imaging has the unique potential to bridge the gap between cellular and molecular biology. Therefore, cryo-EM three-dimensional (3D) reconstruction has been rapidly developed in recent several years and applied widely in life science research to reveal the structures of large macromolecular assemblies and cellular complexes, which is critical to understanding their functions at all scales. Although the technical breakthrough in recent years, for example, the introduction of the direct detection device (DDD) camera and the development of cryo-EM software tools, made the three cryo-EM pioneers share the 2017 Nobel Prize, several bottleneck problems still exist that hamper the further increase of the resolution of single-particle reconstruction and hold back the application of in situ subnanometer structure determination by cryo-tomography. Radiation damage is still the key limiting factor in cryo-EM. In order to minimize the radiation damage and preserve as much resolution as possible, the imaging conditions of a low dose and weak contrast make cryo-EM images extremely noisy with very low signal-to-noise ratios (SNR), generally about 0.1. The high noise will obscure the fine details in cryo-EM images or reconstructed maps. Thus, a method to reduce the level of noise and improve the resolution has become an important issue. In this paper, we systematically reviewed and compared some robust filters in the cryo-EM field of two aspects, single-particle analysis (SPA) and cryo-electron tomography (cryo-ET), and especially studied their applications, such as, 3D reconstruction, visualization, structural analysis, and interpretation. Conventional approaches to noise reduction in cryo-EM imaging include the use of Gaussian, median, and bilateral filters, among other means. A Gaussian filter selects an appropriate filter kernel to conduct spatial convolution with a noisy image. Although noise with larger standard deviations in cryo-EM images can be suppressed and satisfactory performance is achieved in certain cases, this filter also blurs the images and over-smooths small-scale image features. This is especially detrimental when precise quantitative information needs to be extracted. Unlike a Gaussian filter, a median filter is based on the order statistics of the image and selects the median intensity in a window of the adjacent pixels to denoise the image. Although this filter is robust to outliers, it suffers from aliasing problems that possibly result in incorrect information for cryo-EM structure interpretation. A bilateral filter is a nonlinear filter that performs spatial weighted averaging and is more selective in the pixels allowing to contribute to the weighted sum, excluding the high frequency noise from the smoothing process. Thus, this filter can be used to smooth out noise while maintaining the edge details, which is similar to an anisotropic diffusion filter, and distinct from a Gaussian filter but its utility will be limited when the SNR of a cryo-EM image is very low. Generally, spatial filtering methods have the disadvantage of losing image resolution when reducing noise. A wavelet transform can exploit the wavelet's natural ability to separate a signal from noise at multiple image scales to allow for joint resolution in both the spatial and frequency domains, and thus has the potential to outperform existing methods. The modified wavelet shrinkage filter we developed can offer a remarkable improvement in image quality with a good compromise between detail preservation and noise smoothing. We expect that our review study on different filters can provide benefits to cryo-EM applications and the interpretation of biological structures.

Aplicaciones de la Criomicroscopía Electrónica en Producción de Vacunas contra el SARS-CoV-2 / Applications of Electronic Cryomicroscopy in Production of Vaccines against SARS-CoV-2

Este ensayo tiene como objetivo discutir los modelos de propuestas de vacunas contra la SARS-CoV-2 utilizando técnicas de Crio-TEM. Para ello se fundamenta en las ideas de Humberto Fernández-Moran cuando decía, "le llegó su hora a las biomoléculas", su Criomicroscopía Electrónica de Transmisión (Crio-MET) pasó de ser una técnica engorrosa y descriptiva, a ser fácil, estructural y más apropiada para los estudios de las macromoléculas en suspensión a alta resolución cercano al nivel atómico. Después de 50 años las técnicas propuestas y desarrolladas por este ilustre venezolano, son hoy en día vigente y de gran importancia ya que han servido de base a nuevas áreas de estudio como la farmacología molecular basadas en estos principios biofísicos. El Dr. Fernández-Moran logró predecir que "a temperaturas de Helio líquido (-180 °C) y alta velocidad de congelamiento, se podrían analizar estructuras biológicas a alta resolución y en estado dinámico e hidratado libre de cristales de hielo". La Criomicroscopía Electrónica de Transmisión (Crio-MET) es un método biofísico que se emplea para el estudio de biomoléculas y complejos moleculares congelados-hidratados a resoluciones cercanas al nivel atómico. Esta técnica trajo consigo la apertura de nuevas investigaciones en Farmacología Molecular. Su aplicación en el estudio y diseño de anticuerpos moleculares han sido exitosos en la formulación de nuevos fármacos de alta especificad sin la producción de los conocidos efectos secundarios. Hoy en día la Farmacología Molecular ha logrado grandes avances en la posible producción vacunas de enfermedades infecto contagiosas que afectan a la humanidad(AU)

This essay aims to discuss models of SARS-CoV-2 vac-cine proposals using Cryo-TEM techniques. It is based on ideas Humberto Fernández Moran; when he said, "time has come for biomolecules", his Electronic Cryomicroscopy Transmission, (Cryo-TEM), went from being a cumbersome and descriptive technique to being easy, struc-tural and more appropriate for studies of macromolecules in high resolution suspension close to atomic level. After 50 years the techniques proposed and developed by this illustri-ous Venezuelan, are today valid and of great importance since they have served as a basis for new areas of study, such as molecular pharmacology based on these biophysical principles. Dr. Fernandez Moran managed to predict that "at liquid Helium temperatures (-180 ° C) and high freezing speed, biological structures could be analyzed at high resolution and in a dy-namic and hydrated state free of ice crystals." Electronic Cryo-microscopy Transmission, (Cry-oTEM) is a biophysical method that is used to study biomol-ecules and frozen-hydrated molecular complexes at resolu-tions close to the atomic level. This technique brought with it the opening of new research in Molecular Pharmacology. Its application in the study and design of molecular antibodies has been successful in the for-mulation of new high-specific drugs without the production of the known side effects. Today, molecular pharmacology has made great progress in the possible production of vaccines in infectious contagious diseas-es that affect humanity(AU)

EF-hand like Region in the N-terminus of Anoctamin 1 Modulates Channel Activity by Ca²⁺ and Voltage

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca²⁺ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca²⁺ binding. Because the Ca²⁺ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca²⁺ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca²⁺ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca²⁺-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca²⁺ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca²⁺. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca²⁺ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca²⁺ and modulate the activation by Ca²⁺ and voltage.

Single-particle cryo-electron microscopy opens new avenues in structural biology of G protein-coupled receptor / 浙江大学学报·医学版

G protein-coupled receptors(GPCRs)represent the largest class of cell surface receptors,mediating wide range of cellular and physiological processes through their transducers,G proteins and the-arrestins participate in almost all pathological processes. Recent technological advances are revolutionizing the utility of cryo-electron microscopy(cryo-EM),leading to a tremendous progress in the structural studies of biological macromolecules and cryo-EM has played a leading role in the structural biology of GPCR signaling complex. New discoveries of high-resolution threedimensional structures of GPCR signaling complexes based on cryo-EM have emerged vigorously,which depict the common structural characteristics of intermolecular interaction between GPCR and G protein complex-the conformational changes of the transmembrane helix 6 of receptors,and also demonstrate the structural basis of G protein subtype selectivity. Single-particle cryo-EM becomes an efficient tool for identifying the molecular mechanism of receptor-ligand interaction,providing important information for understanding GPCR signaling and the structure-based drug design.

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na/K cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI facilitate Na/K pass through, which was defined as binding-block mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.