Lysosomal Activation Mediated by Endocytosis in J774 Cell Culture Treated with N-Trimethyl Chitosan Nanoparticles.

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 µg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Acetone Detection and Classification as Biomarker of Diabetes Mellitus Using a Quartz Crystal Microbalance Gas Sensor Array.

A gas sensor array was developed and evaluated using four high-frequency quartz crystal microbalance devices (with a 30 MHz resonant frequency in fundamental mode). The QCM devices were coated with ethyl cellulose (EC), polymethylmethacrylate (PMMA), Apiezon L (ApL), and Apiezon T (ApT) sensing films, and deposited by the ultrasonic atomization method. The objective of this research was to propose a non-invasive technique for acetone biomarker detection, which is associated with diabetes mellitus disease. The gas sensor array was exposed to methanol, ethanol, isopropanol, and acetone biomarkers in four different concentrations, corresponding to 1, 5, 10, and 15 µL, at temperature of 22 °C and relative humidity of 20%. These samples were used because human breath contains them and they are used for disease detection. Moreover, the gas sensor responses were analyzed using principal component analysis and discriminant analysis, achieving the classification of the acetone biomarker with a 100% membership percentage when its concentration varies from 327 to 4908 ppm, and its identification from methanol, ethanol, and isopropanol.

Chitosan, Chitosan/IgG-Loaded, and N-Trimethyl Chitosan Chloride Nanoparticles as Potential Adjuvant and Carrier-Delivery Systems.

This work proposes a feasible, reproducible, and low-cost modified method to manufacture chitosan, chitosan/IgG-protein-loaded, and trimethylated chitosan nanoparticles, using microfluidics combined with the microemulsion technique, which differs from the traditional batch process of chitosan-based nanoparticles. The synthesis process consists of generating microreactors of chitosan-based polymer in a poly-dimethylsiloxane ψ-shaped microfluidic device and then crosslinking with sodium tripolyphosphate outside the cell. Transmission electron microscopy demonstrates an improvement in size control and distribution of the solid-shape chitosan nanoparticles (~80 nm) compared to the batch synthesis. Regarding chitosan/IgG-protein-loaded nanoparticles, these presented a core-shell morphology having a diameter of close to 15 nm. Raman and X-ray photoelectron spectroscopies confirmed the ionic crosslinking between the amino groups of chitosan and the phosphate groups of sodium tripolyphosphate in the fabricated samples and the total encapsulation of IgG protein during the fabrication of chitosan/IgG-loaded nanoparticles. Then, an ionic crosslinking and nucleation-diffusion process of chitosan-sodium tripolyphosphate was carried out during the nanoparticle formation, with and without IgG protein loading. The use of N-trimethyl chloride chitosan nanoparticles in vitro on human-keratinocyte-derived cell line HaCaT did not show side effects independently of its concentration from 1 to 10 µg/mL. Therefore, the proposed materials could be used as potential carrier-delivery systems.

Discrimination Improvement of a Gas Sensors' Array Using High-Frequency Quartz Crystal Microbalance Coated with Polymeric Films.

The discrimination improvement of an array of four highly sensitive 30 MHz gas quartz crystal microbalance (QCM) sensors was performed and compared to a similar system based on a 12-MHz QCM. The sensing polymeric films were ethyl cellulose (EC), poly-methyl methacrylate (PMMA), Apiezon L (ApL), and Apiezon T (ApT) and they were coated over the AT-cut QCM devices by the drop casting technique. All the sensors had almost the same film thickness (0.2 µm). The fabricated QCM sensor arrays were exposed to three different concentrations, corresponding to 5, 10, and 15 µL, of ethanol, ethyl acetate, and heptane vapors. The steady state sensor responses were measured in a static system at a temperature of 20 °C and relative humidity of 22%. Our results showed that the 30-MHz sensors have a higher sensitivity than 12-MHz ones (around 5.73 times), independently of the sensing film and measured sample. On the other hand, principal component analysis and discriminant analysis were performed using the raw data of the responses. An improvement of the classification percentage between 12 MHz and 30 MHz sensors was found. However, it was not sufficient, especially for low concentrations. Furthermore, using partition coefficient and discriminant analysis (DA), an improvement of 100% classification of the three samples was achieved for the case of the 30-MHz sensor array.

Reduction of the Measurement Time by the Prediction of the Steady-State Response for Quartz Crystal Microbalance Gas Sensors.

This paper presents a new approach to reduce the measurement time by the prediction of the steady-state using the transient response to ethanol for quartz crystal microbalance gas sensors coated with ethyl cellulose. The experimentally measured response curves were successively fitted using a mathematical model based on the sum of two exponentials with different time constants. The parameters of the model were determined, and the time constants and the magnitude of the steady-state response were analyzed. Even though the time constants did not stabilize well, the parameter corresponding to the magnitude of the steady-state response quickly converged and stabilized after 37 s. Moreover, this calculated parameter was highly correlated with the measured values of the steady-state response, which was measured at five times the longest time constant (83 s) of the model. Therefore, the steady-state response could be predicted with a 55% reduction in the measurement (detection) time.

Antibody-coupled hydroxyapatite nanoparticles as efficient tools for labeling intracellular proteins.

Smart biomaterials for active targeting are a novel way for biosensing, gene and drug delivery, and bioimaging. The functional additives are chosen according to the material carrier characteristics, i.e. the functional mercapto acids of different lengths. In order to identify the target tissue, cell, organ or molecule, the biomaterial must be equipped with a recognizing molecule on its surface. In most cases, semiconductor o metal materials are employed in bioimaging and biosensing applications; in gene and drug delivery area, it is useful to employ porous nanoparticles as carriers. Hydroxyapatite nanoparticles have been proved efficiently in drug delivery. In this work we established a new protocol to obtain smart hydroxyapatite nanoparticles with 3-mercaptopropionic acid and anti-Actin molecules in order to localize actin molecules in cells.

Physicochemical properties of polycaprolactone/collagen/elastin nanofibers fabricated by electrospinning.

Collagen and elastin are the two most abundant proteins in the human body, and as biomaterials offer fascinating properties to composite materials. More detailed investigations including these biomaterials within reinforced composites are still needed. This report describes physicochemical properties of fibers composed of collagen type I, collagen III, elastin and polycaprolactone (PCL). Prior to the electrospinning process, PCL was functionalized through covalent attachment of -NH2 groups by aminolysis reaction with hexamentilendiamine. The fibers were fabricated by electrospinning technique set up with a non-conventional collector. A morphological comparative study was developed at different rations of collagen type I, observing in some cases two populations of fibers. The diameters and morphology were analyzed by SEM, observing a wide array of nanostructures with diameters of ~310 to 693nm. Chemical characterization was assessed by FT-IR spectroscopy and the functionalized PCL was characterized through ninhydrin assay resulting in 0.36mM NH2/mg fiber. Swelling tests were performed for 24h, obtaining 320% for the majority of the fibers indicating morphological stability and good water uptake. In addition, contact angle analysis demonstrated adequate permeability and differences for each system depending mainly upon the type of biopolymer incorporated and the functionalization of PCL, ranging the values from 108° to 17°. Moreover, differential scanning calorimetry results showed a melting temperature (Tm) of ~60°C. The onset degradation temperatures (Td,onset) ranged between 115 and 148°C, and were obtained by thermogravimetric analysis. The local mechanical properties of individual fibers were quantified by atomic force acoustic microscopy. These results propose that the physicochemical and mechanical properties of these scaffolds offer the possibility for enhanced biological activity Thus, they have a great potential as candidate scaffolds in tissue engineering applications.