Toxicity And Mechanism Of Carbon Point In New Type Fluorescence Nanomaterials

Bio-labeling and diagnostic imaging of nanomaterials is an emerging researcharea, especially in exploiting the potential of agents for the targeted diagnosis oftumor. It’s a severe challenge for nanotechnology to make more biocompatiblemolecular probes for tumor diagnosis to enhance the specificity and sensibility oftumor markers by utilizing the unique properties of nanaomaterials such as small sizeeffects, nano-surface effects, quantum effects, special photic, electric and magneticeffects.It is a prerequisite to fulfill biosafety evaluation of the nanomaterials before theyare transferred into pharmaceutical products which might be used clinically. So far,there have been no international guidelines specifically for safety evaluation ofnanomaterials. Toxicity of major nanoscale medicine products has been evaluated inaccordance with the guidelines of conventional toxicity evaluation. Previous studieshave indicated that the size, shape, surface charge, surface modification and otherproperties of nanomaterials could contribute to the metabolism and biological effectsin vivo. In recent years, some nano-pharmaceutical products were withdrawn fromclinic use by FDA due to their new toxicity which was not found in the evaluationaccording to the conventional toxicity test guidelines. The routine toxicity testguidelines are becoming increasingly infeasible for toxicity evaluation of nanobiopharmaceutical products. It is an imminent task for nanotoxicology to enact a setof objective experimental approaches which could real-timely monitor the interactionsof nanoparticles and the body during their exposure in vivo. Much remains to be donefor nanotoxicology.Carbon dots (C-dots) are a novel fluorescent nanoparticles with excellentdecentrality and fluorescent stability, whose diameter are normally less than10nm.The carbon nanomaterials are capable of many excellent fluorescent properties suchas strong fluorescence intensity, anti-photobleaching ability, and continuousexcitation spectrum, which the traditional fluorescent dyes are incapable of.Compared with semiconductor quantum dots, C-dots exhibits excellentbiocompatibility and environmental friendliness because it does not contain anytoxic elements. Meanwhile, the water solubility of C-dots is nice, and their carboxylsurface makes them easy to couple with biological molecules and get functionallymodified. In view of the excellent physical and chemical properties, C-dots arepotentially utilized to prepare tumor diagnosis probes. However, researches in safetyevaluation of C-dots are currently scarce, a complete and systemic understanding oftheir toxic effect on human health is lacking, and more in-depth research in toxicmechanism of C-dots is essential. In this work, we undertook the evaluation of toxicological effects andmechanism of C-dots (a candidate nanomaterials for diagnostic probe labeling),which was supported by the national973Project that involves the application ofnanotechnology to the early diagnosis of gastric cancer warning and critical scientificissues. We finished a systemic research in biological effects and toxic mechanism ofC-dots in accordance with the International Chemical Toxicology Test Guidelinesand the guides for drug evaluation formulated by China Food and DrugAdministration Center. The following experiments were carried out:(1) The characterization of the physicochemical properties of C-dots. Themorphology, particle size, absorption spectra, fluorescence spectra andfluorescence quantum yield and other parameters of C-dots synthesized bynitric oxide method were respectively characterized by atomic forcemicroscopy (AFM), UV-visible spectroscopy, fluorescence spectrometer andother instrument.(2) The evaluation for acute toxicity and subacute toxicity of C-dots. BALB/Cmice and Wistar rats were used as model animals. After a single exposurethrough intravenous injection, the changes of symptoms, food utilization rates,bodyweight, hematologic indicators, serum biochemical indicators andhistopathological lesions of main organs were detected.(3) The genotoxcity of C-dots were evaluated by four combination experiments.Salmonella typhimurium reverse mutation assay (the test Salmonella strainswere TA97, TA98, TA100and TA102), chromosome aberration test, thealkaline single cell gel electrophoresis assay, micronucleus assay in bonemarrow polychromatic erythrocytes were carried out for the detection of genemutation, aberration, DNA injuries induced by C-dots.(4) The effects of C-dots on immune function. Single and repeated exposureswere performed through tail vein injection on BALB/C mice. The effects ofC-dots on macrophage function, proliferative activity of T and B lymphocytes,immune effects and the pathological changes in the related immune organswere analyzed at different time points through lymphocytes proliferation test,types and subsets assay of lymphocytes, neutral red phagocytosis ofmacrophage，detection of the secretion and expression levels of relatedcytokine and analysis of pathological morphology of immune organsThe experimental results indicated:(1) C-dots were synthesized by the method of nitric acid oxidation. Theirphysicochemical properties were characterized. The diameter of C-dotsranged from5to10nm. The rich carboxyl and hydroxyl groups on thesurface of C-dots were confirmed by ultraviolet and infrared spectral analysis,which interpreted the nice water solubility of C-dots in terms of their molecular structure. Fluorescence spectra analysis indicated that C-dots couldemit different fluorescence under various excitation wavelengths. Themaximum excitation wavelength of naked C-dots was about340nm, andC-dots could emit strong blue fluorescence after excitation at this wavelength.The fluorescence quantum yield of the naked C-dots was about0.85%.(2) There were no obvious acute and subacute toxicities found in the modelanimals after single intravenous injection administration. The treated animalsgenerally behaved well, no significant changes were found in the foodutilization rate, bodyweight, hematologic indicators and serum biochemicalindicators. No obvious pathological changes of main organs were observed.(3) The results of genotoxic experiments demonstrated that C-dots were unable toinduce the mutations in Salmonella typhimurium, chromosome aberrationsand DNA damages in CHL cells, micronuclei mutation in bone marrowerythrocytes in treated animals in the administrated dose range of C-dots.Therefore, we concluded that C-dots had no apparent inherent toxicity.(4) The immunotoxicity of C-dots was related to the dose, time and repeatedtimes of administration. When C-dots were exposed single time through thetail vein injection, macrophages were activated and their function wereenhanced. The secretion levels of cytokines IFN-γ were increased. At theinitial stage post exposure, the types, subsets and quantities of lymphocytesonly showed small changes and no significant immune response was detected.However, at the ninth day after administration, C-dots could significantlypromote the proliferation of T lymphocytes, increase the secretion levels ofIFN-γ and TNF-, and enhance the immune effects which were mediated byTh1cells. When C-dots were administrated repeatedly, obvious changes tookplace in the detection indexes, including the promoted proliferation of Tlymphocytes, the increased secretion of IFN-γ and TNF-, and theup-regulated gene expression of related cytokines. What was worthy of notewas that the continuous exposure of C-dots led to the oxidative damage andthe immune balance disruption which might induce the compensablepathological lesions in target organs.The safety evaluation of nanomaterials is an extreme challenging sphere ofresearch. This study is the first one to carry out a systematic and comprehensivetoxicological evaluation on C-dots through a series of experiments at the molecular,cellular and animal levels. The experimental results of general toxicity andgenotoxicity demonstrated that C-dots did not exert significant toxicity in the range ofexposed dosage. Immunotoxicity research showed that C-dots could promote Th1cell-mediated immune response through single injection exposure. It cannot beignored that C-dots could cause the compensable pathological lesions in the target organs after continuous administration. The work undertaken in this paper wouldprovide safety reference for the biomedical and clinical application of C-dots infuture.