| Background:The toxicity evaluation of inorganic nanoparticles has been reported by an increasing number of studies, but toxicity studies concerned with biodegradable nanoparticles, especially the neurotoxicity evaluation, are still limited. Chitosan nanoparticles (CS-NPs) and Tween 80 modified chitosan nanoparticles (TmCS-NPs) are among the most commonly used nanoparitcles as brain vehicles and their potential adverse effects on the CNS are poorly understood. Thus, the neurotoxicity and developmental neurotoxicity (DNT) of these two nanoparticles will be investigated in the present study.Method:CS-NPs and TmCS-NPs with a particle size around 250 nm were prepared based on ionic gelation method and the particle size, zeta-potential, and morphology were characterized. For the neurotoxicity assessments in SD rats, rhodamine B isothiocyanate (RBITC) was adopted to label the chitosan to detect the distribution of the CS-NPs and TmCS-NPs in the brain after intravenous injection. Then rats were exposed to three TmCS-NP concentrations (30mg/kg,10mg/kg,3mg/kg). Consequently, oxidative stress, one of the best-known factors for apoptosis, was assessed in the accumulation regions of the brain. Daily body weight of the rats was measured before tail-vein injection. Histopathological analyses of the brain were also performed to assess the tissue morphology. GFAP expression and NeuN expression were investigated to assess the changes of astrocytes and neurons, respectively. For the DNT assessment using a zebrafish embryo model, the mortality rate, hatching rate, and malformations of embryos exposed to CS-NPs and TmCS-NPs with a series of concentration (5mg/L, 10mg/L,20mg/L,30mg/L,40mg/L,50mg/L) were analyzed firstly. Meanwhile, the LC50 of these two nanoparticles were determined. Afterwards, neurobehavioral assessments and corresponding mechanism studies were performed at a relatively lower exposure concentration,1/2 LC50. Embryos were evaluated at different time points for four types of behavior experiments:spontaneous movement, tactile sensitivity test, free swimming activity, and swimming activity in response to light-dark photoperiod stimulation. And the intracellular ROS levels in embryos, apoptosis analysis with a flow cytometer, apoptosis analysis with AO staining, histopathological examination, TEM of the musce, and whole-mount immunohistochemistry for primary and secondary motor neurons were performed to determine the possible mechanisms underlying nanoparticle-mediated toxicity.Results:CS-NPs and TmCS-NPs were successfully prepared by ionic cross-linking method. The particles size and zeta-potential of CS-NPs were 247 ± 20 nm and 24.4 ± 3.9 nm, respectively. The corresponding values for the TmCS-NPs were 251 ± 15 nm and 26.5 ± 4.2 mV, respectively. Here we reported, for the first time, that TmCS-NPs with a size of 250 nm mostly accumulated in the frontal cortex and cerebellum. After 7d intravenous injection with TmCS-NPs, the body weight of rats remarkably decreased dose-dependently compared with those of the control group. In addition, dose-dependent neuron apoptosis and slight inflammatory response in the frontal cortex, and downregulation of GFAP expression in the cerebellum induced by 7d exposure to TmCS-NPs were detected. These phenomena indicate the potential neurotoxicity of TmCS-NPs. DNT study in zebrafish embryos demonstrated that CS-NPs and TmCS-NPs exposure induced apparent increased mortality rate, deceased hatching rate, and malformations dose-dependently. And the LC50 of CS-NPs was similar to that of the TmCS-NPs. Besides, disturbed neurobehavioral activities, axonal growth of motor neurons, and muscle development were also detected in zebrafish embryos and larvae exposed to CS-NPs and TmCS-NPs at the concentration of 1/2 LC50. In addition, the neurotoxicity assessments using the neurobehavioral analysis, muscle structure analysis, and motor neurons development analysis were more sensitive than developmental toxicity analyses such as mortality and malformation. |
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