USA); bicinchoninic acid (BCA) protein assay kit (Nanjing Keygen Biotech. Co., Ltd, China); learn more interleukin-4 (IL-4) and interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kit (Neobioscience Technology Co., Ltd, Shenzhen, China); concanavalin A (ConA), lipopolysaccharide (LPS) (Sigma-Aldrich Co., St Louis, MO, USA); anti-TNF-α, anti-IL-12,
anti-IFN-γ, anti-IL-4 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA); rabbit anti-β-actin (Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, China). Synthesis and characterization of carbon dots Carbon dots were prepared using the improved nitric acid oxidation method. In the typical experiment, 0.5 g of raw soot was dispersed ultrasonically in acetone solution for 30 min and then centrifugated and dried under vacuum at 80°C. Subsequently, MAPK inhibitor the cleaned soot was refluxed in 25-ml 5 M HNO3 at 120°C for 14 h. The black suspension was cooled down to room temperature and centrifugated at 3,000 rpm for 10 min to remove the unreacted precipitate. The light brown solution was neutralized by Na2CO3 and then dialyzed against Akt inhibitor Millipore pure water (Billerica, MA, USA) for 3 days to remove the salt of sodium through a dialysis membrane with an MW cutoff value of 1,000, affording purified carbon nanoparticles.
After that, further size separation of carbon nanoparticles Histidine ammonia-lyase was performed by stepwise ultrafiltration with NMWL values of 5,000 and 3,000 ultrafiltration membranes using a Millipore stirred ultrafiltration
cell. Finally, the carbon dots were dialyzed with an MCO of 3,000 dialysis membrane. Atomic force microscopy (AFM) images of carbon dots were taken on a MultiMode Nanoscope III, a scanning probe microscopy system (Veeco, Plainview, NY, USA). The samples for AFM were prepared by dropping an aqueous suspension (0.01 mg/ml) of carbon dots on freshly cleaved mica surface and dried under vacuum at 80°C. UV-visible (vis) spectra were measured at 20°C with a Shimadzu UV-2450 UV–vis spectrophotometer (Kyoto, Japan) equipped with a 10-mm quartz cell, where the light path length was 1 cm. Fluorescence spectra were recorded on a HITACHI H FL-4600 spectrofluorimeter (Tokyo Japan). Animal injection, weight measurements, and sample collection Female BALB/c mice (approximately 18 to 22 g), obtained from the Center of Laboratory Animals of Academy of Military Medical Sciences in compliance with the Institutional Animal Care and Use Program Guidelines, were given food and water ad libitum and housed in a 12-h/12-h light/dark cycle. After acclimation, the mice were randomly divided into eight experimental groups, each consisting of ten mice. Before intravenous administration to mice, the carbon dots were well sonicated and diluted in physiologic saline.