MOLECULAR AND BIOMOLECULAR-BASED NANOMATERIALS: TUBULIN AND TAXOL AS MOLECULAR CONSTITUENTS

The new field of protein-based nano-technology takes advantage of the complex interactions between proteins to form unique structures with properties that cannot be achieved with traditional components. Microtubules (MTs), self assembled proteinaceous hollow filaments, offer promise in the development of MT-based nano-systems. The compelling need for the controlled assembly of 3D MT arrays is the fundamental motivation for the first part of this research. We report on the morphology of MTs grown in a crowded environment in the form of high viscosity fluids containing agarose and a novel process that enables the assembly of MTs supported by gel-based 3D scaffolds. Our research on MTs and their interaction with other molecules lead us to discover extraordinary spherulitic structures that changed the course of the project. The novel subject situate us into a complicated dilemma that question the nature of MT asters reported in experiments carried out in cells. The second part of this research is focused in the crystallization ofTaxol, a MT stabilizing molecule used as anti-cancer drug. It was confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated with fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Furthermore, the crystallization of Taxol was studied in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We demonstrated that tubulin is able to heterogeneously nucleate Taxol spherulites. To explain the formation of tubulin-Taxol nuclei a new, secondary Taxol-binding site within the tubulin heterodimer is suggested. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters. Thus, we are confirming the need for careful interpretation of fluorescence microscopy observations of MT structures when large concentrations of Taxol are used as stabilizing agent in cells

Plasma and chemical treatments of zirconia surface for cell response improvement: A comparative study

Zirconia is an inert implantable biomaterial with poor or null cell response. To enhance this condition of inertness, surface modification can be used to induce bioactivity improving the implant-tissue interaction. For bioactivation, a zirconia surface was modified by means of two treatments, one with argon plasma and the other with calcium and phosphate ions in addition to plasma. In vitro tests were performed to determine cell response on both surfaces and on a control. The results showed similar evidence of bioactivation in the treated samples. The novelty of this paper lies in the cellular response studies carried out in zirconia treated surfaces with two different bioactivation processes and its comparative evaluation, including cell viability, morphology and spreading. To our knowledge, no prior studies have reported these experiments, which may have many applications in the field of biomaterials

Thermosensitive hydrogel for in situ-controlled methotrexate delivery

Abstract: Methotrexate (MTX) is widely used for the treatment of various types of cancer; however, it has drawbacks such as low solubility, lack of selectivity, premature degradation, and side effects. To solve these weaknesses, a hydrogel with the ability to contain and release MTX under physiological conditionswithout burst release was synthesized. The hydrogel was fabricated with a poly(ɛ-caprolactone)-bpoly( ethylene glycol)-b-poly(ɛ-caprolactone) (PCL–PEG–PCL) triblock copolymer, synthesized by ring-opening polymerization. The characterizations by proton nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectrometry confirmed the copolymer assembly, whereas the molecular weight analysis validated the PCL2000–PEG1000–PCL2000 structure. The copolymer aqueous solution exhibited sol–gel phase transition at 37°C and injection capacity. The hydrogel supported a load of 1,000 μg MTX·mL−1, showing a gradual and sustained release profile of the drug for 14 days, with a delivery up to 92% at pH 6.7. The cytotoxicity of the MTX-loaded hydrogel was performed by the methyl thiazole tetrazolium assay, showing a mean inhibitory concentration of 50% of MCF-7 cells (IC50) at 43 μg MTX·mL−1

pH-responsive polymer micelles for methotrexate delivery at tumor microenvironments

Methotrexate (MTX) anticancer drug was successfully loaded and released in a controlled manner from polymer micelles made of a diblock copolymer of poly(monomethoxy ethylene glycol)-b-poly(ε-caprolactone) (mPEG-PCL). The empty and MTX-loaded micelles (MTX/mPEG-PCL) were characterized by electron microscopy. The drug release dependence upon pH 5.4, 6.5, and 7.4 for 30 days was proven and characterized by UV-Vis spectroscopy. The cytotoxic effect of MTX/ mPEG-PCL micelles on MCF-7 breast cancer cells was evaluated through an MTT assay. The morphological analysis indicated the successful formation of micelles of 76 and 131 nm for empty and MTX-loaded micelles, respectively. An encapsulation efficiency of 70.2% and a loading capacity of 8.8% were obtained. The in vitro release of MTX showed a gradual and sustained profile over 22 days, with a clear trend to much higher release at acidic pH (80 and 90% for pH 6.7 and 5.5, respectively). The MTX/mPEG-PCL micelles showed an IC50 of MCF-7 cells at 30 µg mL−1 . The results suggested that MTX/mPEG-PCL could be a promising drug delivery system for cancer treatmen

Bioactivation of zirconia surface with laminin protein coating via plasma etching and chemical modification

Surface modification offers a promising alternative to provide bioactivity to implanted inert biomaterials, improving their integration and performance with living tissues. In this contribution, zirconia has been used as a substrate to investigate the biofunctionalization process, designed to add surface bioactivity to a bioinert biomaterial. We intended to attach laminin-5, known for its ability to bind epithelial cells in soft tissue, to the surface of the extracellular matrix protein. First, the zirconia surface was etched and activated with argon plasma and subsequently it was chemically functionalized with calcium and phosphate ions. Zirconia surface activation was monitored by means of a wettability test, whereas functionalization with calcium and phosphate ions was evaluated by confocal Raman microscopy, Z-potential and X-ray Photoelectron Spectroscopy (XPS). The binding of laminin-5 protein to the zirconia surface was carried out by means of adsorption and confirmed by XPS. Then, we used SEM and AFM to observe a homogeneous covering of globular protein over the zirconia surface. Furthermore, epithelial cell response over zirconia surfaces was assayed to show that biofunctionalized surfaces enhance cell adhesion to a greater extent than substrates without protein coating. Our results indicate how the zirconia surface can be modified using argon plasma, in order to enable its bioactivation with the laminin-5 protein

Síntesis y caracterización de micelas poliméricas de mPEG-PCL para sistemas de suministro de fármacos: rescatando un proceso sencillo

El propósito de este estudio era reproducir un procedimiento no común y flexible para la formación de micelas de copolímeros. Aunque, estas estructuras han sido consideradas como uno de los portadores más potenciales en el campo de la administración de fármacos, los métodos más comunes utilizados para su reproducción implican el uso de varios disolventes orgánicos tóxicos, y en general, se requieren tiempos largos para asegurar la formación de micelas. Por lo tanto, debido a la necesidad de mejorar la forma de obtener micelas, que encontramos en la literatura un proceso de formación de micelas que tiene ventajas notables sobre los tradicionales. El copolímero bloque utilizado fue poli metoxi (etilenglicol) -poli (ԑ-caprolactona) mPEG-PCL y se obtiene por polimerización catiónica de apertura de anillo. Las técnica de caracterización utilizadas para verificar la copolimerización fueron la Reflectancia Total Atenuada de Espectroscopia Infrarroja con Transformadas de Fourier (ATR-FTIR), y la Resonancia Magnética Nuclear (HNMR). Para la morfología de las micelas se empleó Microscopía Electrónica de Transmisión (TEM), Microscopía Electrónica de Barrido (SEM) y las técnicas de Microscopía de Fuerza Atómica (AFM). Dispersión de Luz Dinámica (DLS) fueron utilizadas para determinar el tamaño y la distribución de las micelas; y las pruebas de estabilidad y la Cromatografía de Permeación en Gel (GPC) se utilizó para determinar los pesos moleculares

Influence of Rare Earths Additions on the Microstructure and Hardness of Heat-treated Nanostructured Superalloy Inconel 718

The superalloy Inconel 718 is greatly employed in high-temperature applications due to its principal characteristics: high resistance to oxidation and excellent mechanical performance at elevated temperatures; hence, its main uses are in aeronautics and aerospace engines, nuclear power generation and petrochemical industries

Effect of Ni additions and hot deformation on precipitation behavior and hardness in AleSieMg aged alloys

Additions of 1e2 Ni (wt. %) and hot deformation on the microstructure and hardness of A356 aged alloy were studied. The results show that the addition of this element generates the formation of Ni31Si12 and NiFe phases with high thermal stability. In addition, the presence of Ni into the Al matrix and precipitates of the second phase affect the precipitation kinetics, transformation sequence, growth rate, and slow loss of hardness in the system after reaching the maximum values. For example, the Ni additions contribute to generating the coexistence of b" precipitate and GP-I zones. Of all the strengthening mechanisms present, hardening by precipitation is the most influential in the hardnes

Effect of Hot Isostatic Pressing and Rare-Earth Elements Addition on the Microstructure and Hardness on Inconel 718

Inconel 718 superalloy is highly employed in high-temperature applications; its principal properties are high resistance to oxidation and excellent mechanical performance. Such alloys are generally used in aeronautics and aerospace engines, power generation, and the chemical industry. Recent studies on the influence of rare-earth elements as rhenium, hafnium, tantalum, niobium, and ruthenium have enlarged to develop superalloys with higher microstructural properties and mechanical performance. Also, significant effects on microstructural and mechanical properties because of cerium and yttrium additions have been reported due to an increase in the lattice mismatch and the grain boundaries, furthermore, promotes modifications in carbides and eutectic phases