Impact of Graphene Oxide and Boron Nitride nanosheets on macrophages in vitro and in vivo in the context of the pulmonary exposure

Abstract

Nanotechnology interest is growing with each passing year, two prominent 2D nanomaterials are graphene oxide (GO) and hexagonal boron nitride (hBN). However, with such applications questions of safety must be answered, as environmental release will become an increasing occurrence. This will in turn increase the risk of environmental exposure occurring, with inhalation being a major route of exposure. Due to nanomaterials size and properties they can travel deep into the lungs, such as the alveolar region. Within the alveolar region any potential inflammation or toxicity could cause permanent damage to the surrounding respiratory tissues. Within the PhD the risk of GO and hBN were investigated, specifically focusing on macrophages being a predominant immune cell within the respiratory system. The work was split into 3 main areas; the role of lung surfactant biocoronation, in vitro direct macrophage/nanomaterial interactions and finally in vivo response from pulmonary exposure. For the first area, two surfactant models were developed and characterised with techniques such as AFM, DLS and ELS. Characterisation indicated that, with biocoronation, zeta potential became less negative but increased height and aggregation. For the second area, bone marrow derived macrophages (BMDM) and immortalised THP-1 macrophages were exposed to GO of different lateral sizes and hBN of one lateral size in their bare or biocoronated forms for one or multiple exposures. The in vitro endpoints were investigated with techniques such as ELISA, RT-qPCR, Raman and confocal and bio assays. The in vitro exposures showed that for both primary and immortalised macrophages, GO did not induce any toxicity or inflammation, while hBN induced inflammation in primary macrophages, but not toxicity. Biocoronation did not significantly alter the responses to either nanomaterial. Innate immune memory was also investigated due to its potential as a chronic safety risk, but limited evidence was found of its induction. For the final area, in vivo analysis with C57BL/6 mice was conducted. Pulmonary tissue was exposed via intratracheal instillation for 1 to 3 exposures of 24 hours. Flow cytometry, multiplex RT-qPCR and H&E staining indicated that exposure to GO induced some minor inflammation but rapidly resolved, while hBN exposure induced no inflammation or toxicity regardless of the exposure. Staining of the tissue by H&E and Raman mapping indicated that GO and hBN were still within the pulmonary environment for each exposure and engulfed by innate cells. An additional in vivo investigation for innate memory found no significant evidence of induction. Overall the findings indicate that exposure to GO or hBN induced either no or limited inflammation, and no toxicity or innate memory. Lateral size and surface biocoronation was shown to have minimal impact on interactions. In all the data collected provides good indications for the potential risk from exposure to sterile 2D engineered nanomaterials, further investigations will still be required for a full safety profile.

Date of Award	1 Jan 1824
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew MacDonald (Supervisor) & Cyrill Bussy (Supervisor)