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Transport of Engineered Nanomaterials across the blood-brain-barrier

Periodic Reporting for period 1 - NanoBBB (Transport of Engineered Nanomaterials across the blood-brain-barrier)

Reporting period: 2018-04-01 to 2020-03-31

Over the last two decades, engineered nanomaterials (ENMs) have been used increasingly in novel technological, biomedical and consumer product applications. However, along with their ground-breaking potential, the unique properties of ENMs have also raised concerns about the potential for unintended consequences on environmental safety and human health. To date, potential toxicity mechanisms of ENMs are not well understood, and there are particular concerns for the potential of ENMs to elicit neurotoxicity. A possible mechanism for such an effect may be the result of ENMs crossing the blood brain barrier (BBB) and then directly or indirectly acting on the central nervous system (CNS). Currently, two key questions remain unanswered regarding the neurotoxicity of ENMs: (1) Can ENMs cross the BBB? Several in vitro and in vivo studies have suggested at least some ENMs can move across the BBB; the range of ENMs capable of such activity and the specific conditions required are still poorly understood, although it is recognised that the capacity of ENMs to cross the BBB is highly related to their physicochemical properties. The literature to date remains fragmented and inconsistent, while ENMs in many studies are not well characterized, making comparisons across the literature highly unreliable. A systematic investigation on the BBB-penetrating ability and its association with the intrinsic properties of ENMs is urgently needed. (2) What is the fate of ENMs within and beyond the BBB? i.e. the deposition, translocation, and transformation of ENMs during and after crossing the BBB. To date, this question has not been addressed well, primarily due to the difficulties in identification and localisation of ENMs in the complex brain environment - even more so for ENMs with high elemental backgrounds (e.g. C-based and Fe-based ENMs). Thus, novel approaches are urgently needed to enable a breakthrough in our understanding of ENM ability to cross the BBB and trace their path beyond. Project NanoBBB proposed combining novel labelling techniques with advanced in vitro methods coupled with in vivo experiments, enabling a uniquely new and systematic approach to elucidate the behaviour and fate of ENMs in the brain and contribute to their future safer design by avoiding any characteristics that may facilitate crossing the BBB.
The overall workflow of this project was: a. Synthesis of ENMs. b. Characterization and stability-testing of ENMs. c. Establishment of an in vitro BBB model. d. Assessment of the transport and distribution of ENMs. e. In vivo validation. The original plan was to synthesize and label a broad library of ENMs, including Fe3O4, CeO2, and GO. However, given budgetary and time limitations, it was decided to focus on Fe3O4, ZnO, CeO2, Ag. The background level of these elements in cells is negligible, so there is no necessity to be labelled. In total, 10 nanoparticles was synthesized. Fe3O4 with two different sizes (Fe3O4 NPs-18nm, Fe3O4 NPs-35nm), CeO2 with three sizes (CeO2 NPs-3nm, CeO2 NPs-7nm, CeO2 NPs-25nm), Ag with four shapes (Spherical Ag nanoparticle (Ag NS), disc-like Ag nanoparticle (Ag ND), rod-like Ag nanoparticle (Ag NR), Ag nanowires (Ag NW)), and ZnO (ZnO NPs). All the particles were characterized using DLS and TEM for the size measurement. Then, an in vitro BBB model displaying key BBB features and functions was successfully set up and validated. It can be used for transport studies. Next, the nanoparticle transport across the BBB was tested using the in vitro BBB model, and analysed by ICP-MS and SP-ICPMS combined with synchrotron radiation-based X-ray absorption fine structure spectroscopy (XAFS). 8 of 10 particles transport across the BBB: CeO2 NPs-3nm, CeO2 NPs-7nm, CeO2 NPs-25nm, Ag NS, Ag ND, Fe3O4 NPs-18nm, Fe3O4 NPs-35nm, and ZnO NPs. For CeO2 NPs-3nm, CeO2 NPs-7nm, CeO2 NPs-25nm, Ag NS, Fe3O4 NPs-18nm, and Fe3O4 NPs-35nm, most of transported elements are in the form of particle. For Ag ND and ZnO, ion accounted for the most of the elements. For Ag NS and Ag NW samples, further LCF analysis of the NEXAFS spectra using standard references compounds showed that the Ag were mainly presented as particulate Ag, with a small proportion as Ag complex (Ag-cystine).

In total, seven papers were generated under this project, published in Nature Protocols, Nano Today, Nanoscale, Small, Environmental Science & Technology, , Environmental International, Environmental Science & Technology Letters. Results have been also disseminated in several international conference including 13th International Conference On The Environmental Effects Of Nanoparticles And Nanomaterials (ICEENN 2018, September 5-8, Duke University, USA), SOT conference 2018 (March 11-15, San Antonio Texas, USA), China Nano 2019 (July 22-26, 2019, Beijing, China), The 10th National Conference on Environmental Chemistry 2019 (15-19 August, 2019, Tianjin, China).
Firstly, the in vitro BBB model developed in this project can be also used to screen nanomedicine given the enormous potential of engineered nanomaterials for use in nanomedicine. Secondly, this study for the first time combines a cutting-edge detecting technique, single particle ICP-MS, with synchrotron radiation-based X-ray absorption fine structure spectroscopy to systematically study the behaviour and fate of certain ENMs in the brain, using an in vitro blood brain barrier model.
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