Study On The Factors Affecting The Immunological Safety Of Carbon Nanotubes

Carbon nanotubes (CNTs) are widely applied in nanotechnology, electronics, optics and other fields within material science and technology owning to their unique physical, electrical and chemical properties. Due to differences in the choice of CNTs and testing conditions, previous studies yielded inconsistent results. The present study was designed to investigate the effects of CNTs with varied diameters, lengths and functionalizations on the immune system to take a comprehensive view of immunotoxicity of CNTs.21 kinds of CNTs have been studied, which were divided into 7 groups. Each group has 3 kinds of CNTs with the same diameter and length but by different modified, including pristine CNTs (P-CNTs), hydroxylation of carbon nanotubes (CNTs-OH) and carboxylic carbon nanotubes (CNTs-COOH). In vitro studies, PBMCs were respectively exposed to 25,50,100 or 200 μg/mL of 21 kinds of CNTs for 12h,24h,48h and 72h, then judgment of PBMCs cytotoxicity of CNTs by measuring cell viability, the amount of LDH and ROS production. At the same time, the supernatant in 24h to be determine the effect of CNTs on PBMCs inflammatory and immune system stimulation by ELISA. The normal human fresh serum was incubated with 21 kinds of CNTs with different concentrations of 25,50,100 or 200 μg/mL. After 1h, through detection of serum complement factors contents to observe the effects of CNTs on complement activation and acute inflammation. In vivo, all CNTs were administered intratracheally (1 or 10 mg/kg) to Balb/c mice. At 1,3 and 7 days after CNTs instillation, the animals were sacrificed with sodium pentobarbital, and the organs, including the lungs and spleens, were removed to measure organ coefficients. We examed pro-fibrogenic cytokines and pro-inflammatory cytokine (including TNF-α, HYP, PDGF, TGF-b, IL-1(3 and IL-6) in the supernate of broncho alveolar lavage fluid (BALF) and lung homogenate, and the production of 14 cytokines in the mice peripheral blood at 1,3 and 7 days. Complement factors of C1q, MASP and C3 in the sera of mice were detected at 1,7 and 28 days.Using human PBMCs, our in vitro data showed clear cytotoxic property of all CNTs when applied at the same dosage, irrespective of their diameters, lengths and functionalizations. In vitro results demonstrate clear cytotoxic property of all CNTs indicate that SWNTs with small diameters were the most potent in inducing oxidative toxicity, despite causing only moderate cell membrane damage. It is also noteworthy that thin-walled CNTs appeared significantly more toxic than thicker-walled CNTs, while CNTs with short length were more toxic compared to longer CNTs. The CNTs-induced cytotoxicity is likely resulted from ROS-induced oxidative stress caused by cellular internalization of CNTs.Regarding potential respiratory hazard of CNTs, we exposed mice to different types of CNTs by intratracheal instillation. We observed increased organ coefficieNTs in all CNTs-treated groups than controls, confirming an immunogenic property of CNTs in vivo. P-CNTs are likely to cause direct toxicity whereas the carboxyl (-COOH) functionalized CNTs are prone to induce indirect immunotoxicity. CNTs stimulate the production of inflammatory mediators, including IL-1β, TNF-α, TGF-β and PDGF. F-CNTs exhibited lower inflammatory production compared with P-CNTs when applied at low concentration. When administrated at high dosage, both CNTs-COOH and CNTs-OH, induced pro-inflammatory cytokine production, confirming a positive correlation between CNTs and pulmonary inflammation. CNTs could decrease pulmonary SOD while increase MDA. P-CNTs showed more significant effects on oxidative stress when applied at a low dosage, whereas CNTs-OH and CNTs-COOH showed more significant effects when high dosage was used. The present study confirmed that nearly all CNTs elicited significant inflammatory responses in vivo, shown as increased levels of IL-1β,IL-6 and TNF-α. Compared to functionalized CNTs-COOH and CNTs-OH, almost all types of P-CNTs, including P-SWNTs and P-MWNTs, induced more robust immune responses following instillation. IL-2 can stimulate the growth of T cells, and we found that P-CNTs and CNTs-COOH significantly promoted IL-2 secretion, which may explain increased spleen coefficients following CNTs-COOH instillation. Production of Th2-related cytokines (IL-4, IL-5 and IL-10) and Th17-type cytokines (IL-17A and IL-22) was also stimulated by P-CNTs and CNTs-COOH in vivo. In addition, hydroxyl (-OH) functionalized CNTs elicited weaker cytokine secretion compared to P-CNTs. CNTs can cause the activation of complement in a short time, and after 28 days, the concentration of complement factors gradually returned to normal level. The minimal SWNTs group was able to induce macrophages to synthesize complement proteins, and the rate of C3 synthesis induced by three kinds of CNTs was determined by the rate at which they entered the cell.The type, diameter, length and functionalization of CNTs were all important parameters affecting CNTs immunotoxicity. We examined cytotoxicity and immune responses of a comprehensive selection of CNTs with varied diameters, lengths and functionalizations. Despite some inconsistency regarding the toxicities of CNTs in vitro and in vivo, P-CNTs exhibited the most potent immunogenic property. In contrast, CNTs-COOH shows weaker effect on immune responses, especially Thl-mediated inflammatory responses. Considering the variety of clinical applications based on CNTs, including gene/drug carriers or vaccine adjuvants, immunotoxicity of different CNTs will have to be taken into consideration when making the selection of CNTs.