A Novel Method for Carbon Nanotube Functionalization Using Immobilized Candida antarctica Lipase.

Carbon nanotubes (CNTs) have been proposed as nanovehicles for drug or antigen delivery since they can be functionalized with different biomolecules. For this purpose, different types of molecules have been chemically bonded to CNTs; however, this method has low efficiency and generates solvent waste. Candida antarctica lipase is an enzyme that, in an organic solvent, can bind a carboxylic to a hydroxyl group by esterase activity. The objective of this work was to functionalize purified CNTs with insulin as a protein model using an immobilized lipase of Candida antarctica to develop a sustainable functionalization method with high protein attachment. The functionalized CNTs were characterized by scanning electron microscope (SEM), Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic functionalization of insulin on the surface of the CNTs was found to have an efficiency of 21%, which is higher in conversion and greener than previously reported by the diimide-activated amidation method. These results suggest that enzymatic esterification is a convenient and efficient method for CNT functionalization with proteins. Moreover, this functionalization method can be used to enhance the cellular-specific release of proteins by lysosomal esterases.

Safe Administration of Carbon Nanotubes by Intravenous Pathway in BALB/c Mice.

Carbon nanotubes (CNTs) are nanomaterials with multiple possible uses as drug carriers or in nanovaccine development. However, the toxicity of CNTs administered intravenously in in vivo models has not been fully described to date. This work aimed to evaluate the toxic effect of pristine multi-walled CNTs (UP-CNTs), purified (P-CNTs), or CNTs functionalized with fluorescein isothiocyanate (FITC-CNTs) administered by intravenous injection in BALB/c mice. Biochemical and histopathological parameters were analyzed at 1, 14, 29, and 60 days post-exposure. Pristine CNTs were the most toxic nanoparticles in comparison with P-CNTs or FITC-CNTs, increasing serum AST (≈ 180%), ALT (≈ 300%), and LDH (≈ 200%) levels at one day post-exposure. The urea/creatinine ratio suggested pre-renal injury at the 14th day accompanied of extensive lesions in kidneys, lungs, and liver. Biochemical and histological findings in mice exposed to P-CNTs had not significant differences compared to the controls. A lower toxic effect was detected in animals exposed to FITC-CNTs which was attributable to FITC toxicity. These results demonstrate that the purification process of CNTs reduces in vivo toxicity, and that toxicity in functionalized CNTs is dependent on the functionalized compound. Therefore, P-CNTs are postulated as potential candidates for safe biomedical applications using an intravenous pathway.

Gut-microbiota-on-a-chip: an enabling field for physiological research.

Overwhelming scientific evidence today confirms that the gut microbiota is a central player in human health. Knowledge about interactions between human gut microbiota and human health has evolved rapidly in the last decade, based on experimental work involving analysis of human fecal samples or animal models (mainly rodents). A more detailed and cost-effective description of this interplay is now being enabled by the use of in vitro systems (i.e., gut-microbiota-on-chip systems) that recapitulate key aspects of the interaction between microbiota and human cells. Here, we review recent examples of the design and use of pioneering on-chip platforms for the study of the cross-talk between representative members of human microbiota and human microtissues. In these systems, the combined use of state-of-the-art microfluidics, biomaterials, cell culture techniques, classical microbiology, and a touch of genetic expression profiling have converged for the development of gut-on-chip platforms capable of recreating key features of the interplay between human microbiota and host human tissues. We foresee that the integration of novel microfabrication techniques and stem cell technologies will further accelerate the development of more complex and physiologically relevant microbiota-on-chip platforms. In turn, this will foster the faster acquisition of knowledge regarding human microbiota and will enable important advances in the understanding of how to control or prevent disease.

Cytotoxicity of protein-carbon nanotubes on J774 macrophages is a functionalization grade-dependent effect.

Carbon nanotubes (CNTs) are used as carriers in medicine due to their ability to be functionalized with chemical substances. However, cytotoxicity analysis is required prior to use for in vivo models. The aim of this study was to evaluate the cytotoxic effect of CNTs functionalized with a 46 kDa surface protein from Entamoeba histolytica (P46-CNTs) on J774A macrophages. With this purpose, CNTs were synthesized by spray pyrolysis and purified (P-CNTs) using sonication for 48 h. A 46 kDa protein, with a 4.6-5.4 pI range, was isolated from E. histolytica HM1:IMSS strain trophozoites using an OFFGEL system. The P-CNTs were functionalized with the purified 46 kDa protein, classified according to their degree of functionalization, and characterized by Raman and Infrared spectroscopy. In vitro cytotoxicity was evaluated by MTT, apoptosis, and morphological assays. The results demonstrated that P46-CNTs exhibited cytotoxicity dependent upon the functionalized grade. Contrary to what was expected, P46-CNTs with a high grade of functionalization were more toxic to J774 macrophages than P46-CNTs with a low grade of functionalization, than P-CNTs, and had a similar level of toxicity as UP-CNT. This suggests that the nature of the functionalized protein plays a key role in the cytotoxicity of these nanoparticles.

Cytotoxicity of functionalized carbon nanotubes in J774A macrophages.

Cytotoxicity of carbon nanotubes (CNTs) is a prime concern for its use as antigen carriers. Here we evaluated the cytotoxic effect of unpurified (UP-CNTs), purified (P-CNTs), fluorescein isothiocyanate-functionalized (FITC-CNTs), and Entamoeba histolytica 220-kDa lectin-functionalized CNTs (L220-CNTs) in J774A macrophage (MOs) cell line. Cell viability and apoptosis were analyzed by MTT and TUNEL assays, respectively. Cyclooxygenase-2 (COX-2) was analyzed by reverse transcription-polymerase chain reaction. Cytotoxicity at 6.0 mg/L was higher with UP-CNTs > P-CNTs > FITC-CNTs, showing a decrease in cell viability and an increase in apoptosis. In contrast, MOs interacted with L220-CNTs showed an increase in cell viability without signs of apoptosis. Although UP-CNTs and P-CNTs exhibited COX-2 induction with 6.0 mg/L, functionalized CNTs were able to induce COX-2 at concentrations as low as 0.06 mg/L. These results suggest that functionalization decreases toxicity, and that L220-CNTs may be an excellent candidate for the production of a nanovaccine against amebiasis.