Silver nanoparticles (AgNPs), as a broad-spectrum antimicrobial agent, have been widely applied in numerous sectors, including personal care products, dietary supplements, and medical supplies. Inevitably, AgNPs can be released into the environment during the manufacturing, usage, and disposal of AgNP-based products. These arouse emerging concerns regarding to the risks to the environmental organisms and human health. After entering into the environment and organisms, AgNPs may undergo different morphology and chemical transformations as well as biotransformations, and their highly dynamic property can lead to the co-existence of different Ag species. While the toxicities induced by different silver species are closely related to their morphology, the clarification of the toxicity by silver species is still remaining a challenge, due to the lack of a versatile analytical technique to quantitatively discriminate different Ag species. Thus, developing a robust method to separate and quantify different Ag species in complex biological matrices is of paramount importance for understanding of the biodistribution and transformation behavior of Ag species. In addition, based on the toxicology methods, the silver transformation behaviors can hopefully be linked to their corresponding toxicity, which opens new opportunities for shedding new light on the toxic mechanism and safety assessments of Ag species.
This dissertation focuses on the characterization and quantification of Ag species in environmental organism and mammals, as well as clarifing their biodistribution, transformation and toxic effects. Coupling the tissue digestion and Ag species extraction by tetramethylammonium hydroxide (TMAH) with size exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICP-MS), the methods for separation, quantification and characterization of different Ag species in microorganisms and rats were developed for the investigation of the biodistribution and transformation of Ag species in vivo. Furthermore, combining conventional toxicity tests, we discussed the toxic effects from different Ag species based on their biodistribution and transformation behavior to study the toxic mechanisms of different species in vivo.
Firstly, a method based on the SEC-ICP-MS system was proposed for simultaneous separation and quantification of various Ag species including particulate Ag, complexed Ag(I) and free Ag+ in Escherichia coli (E.coli). To completely eliminate the interferences of cell wall-adsorbed Ag to the determination of the internalized Ag in E.coli, the lysozyme was employed to remove the cell wall from E.coli and then obtain its protoplasmic cell. After that, into the cell lysates was added the surfactant FL-70 to stabilize AgNPs, and then TMAH for the cell matrices digestion and various Ag species extraction. Finally, SEC-ICP-MS was utilized to realize the separation and quantification of different Ag species in E.coli. The proposed method provided low detection limites with 3 ng/mL for ionic Ag and AgNPs with nominal sizes of 10 nm, 20 nm and 30 nm. The determined recoveries and precisions of different Ag species in E.coli cells at three spiked levels were 82.19%-115.8% and < 0.9%, respectively, suggesting the practicality of this proposed method for the separation and quantification of Ag species in E.coli. In addition, it was found that the ionic Ag in E.coli existed in the form of Ag(I)-biomolecule complexes. However, within a certain amount of E.coli (107 CFU/mL), the limited amount of biomolecules available for complexing with Ag+ set up an upper limit for the amount of complexed Ag(I) (2 μg/mL), and the Ag+ at a spiked level above this limit would exist in the form of free Ag+. This method provided an efficient strategy for the investigation of Ag biodistribution and transformation in E.coli.
Secondly, the transformation behavior and the related toxicity of different Ag species in E.coli were studied. The E.coli mortalities (EC10, EC50 and EC90) in treatment with Ag+ and AgNPs with nominal sizes of 10 nm and 30 nm were determined by viable plate counts, and then the quantification and speciation of Ag species within E.coli under these three doses were performed based on the proposed method described above. More importantly, since SEC-ICP-MS system is applicable for the quantification and speciation of Ag species in the extracellular matrix, the contents of the cell wall-adsorbed Ag could be obtained by subtracting the contents of the intracellular and extracellular Ag from the total exposure dose of Ag, and therefore the biodistribution of Ag species could be profiled. The results revealed that different doses of Ag+ and different sized AgNPs exposed to E.coli showed similar amplitude of heterogeneity, i.e, the intracellular uptake Ag varied from 5.98% to 15.21% of the dose and the cell wall-bound Ag from 25.13% to 64.43% of the dose, demonstrating that both of the cell wall-adsorption and cellular internalization of Ag species existed during the exposure. Furthermore, after exposure to different sized AgNPs, it was found that AgNP could be internalized by E.coli and further release Ag+; and the released Ag+ increased with the decrease of the AgNP particule size. Although the dissolved Ag(I) concentrations in E.coli exposed to different sized AgNPs were higher than that after Ag+ exposure, they showed almost the same E.coli mortality, demonstrating that the toxicity induced by the Ag+ released from AgNP was lower than that by the initial exposed Ag+ to E.coli. The results might be attributed to the complexation of the released Ag+ from AgNP with the biomolecules, which led to the detoxification. Besides, due to the dose-response of different Ag species, the free Ag+ was not likely to be existed in the living E.coli cells unless the total ionic Ag is > 2 μg/mL.
In the third part, another method for simultaneous separation, mass quantification and size characterization of different Ag species in various organs of mammals was established. The tissue digestion and Ag species extraction by TMAH were performed by spiking Ag species into swine liver. The digestion conditions (digestion time and TMAH concentration) were optimized, and then both particulate and ionic silver were extracted in their original forms from the organs and analyzed by SEC-ICP-MS. It was reported that when AgNPs enter into physiological fluids, biomolecules tend to adsorb onto the particle surface to form a biomolecule corona (BC) and this AgNP would exist in the form of AgNP@BCs. While the silver mass concentrations were quantified by ICP-MS with a detection limit of 0.1 μg/g, the effective diameter of AgNP@BCs was determined based on the retention time in SEC separation with size discrimination of 0.6–3.3 nm. More importantly, we found that the BC thickness of AgNP@BCs is core size independent, and a linear correlation was found between the effective diameter and core diameter of AgNP@BCs in extracted tissues, which was used to calibrate the core diameter with standard deviations in the range of 0.2–1.1 nm. The utility of this strategy was demonstrated through validation to various rat organs including liver, heart, spleen, lung, kidney and brain, providing an efficient approach for separation, mass quantification and size characterization of the core-biocorona structure and dissolved species of AgNPs in different organs in rats.
While the highly dynamic properties of AgNPs lead to coexposure to particulate and ionic silver, their combined effects on biodistribution, transformation and toxicity in vivo are still unclear. Herein, after intravenous exposure to either Ag+, AgNPs, or a mixture of Ag+ and AgNPs, the distribution of different silver species in rat organs was quantitatively determined based the proposed method as described above, and the corresponding toxicities were profiled by combining the Ag species biodistribution with histopathological, biochemical and immunohistochemical analyses to elucidate the combined effects of particulate and ionic Ag. Most of the accumulated silver existed in ionic form in all organs, only a small fraction of AgNPs was detected in liver and lung, and the AgNPs taken up in organs were either dissolved completely in a short time or maintained in their original core size in 3 days postadministration, and completely transformed into Ag(I) at day 5. While there was a negligible combined effect on the uptake or redistribution of different silver species, the coexistence of Ag+ accelerated the dissolution of administered AgNPs by the remarkable overexpression of metallothioneins, and the presence of AgNPs could alleviate the Ag+-induced toxicity.