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for a wide variety of applications. Biomedical fields have experienced important new achievements with technological breakthroughs obtained from 2D materials with singular properties. Boron nitride nanosheets are a novel 2D layered material comprised of a hexagonal boron nitride network (BN) with interesting intrinsic properties, including resistance to oxidation, extreme mechanical hardness, good thermal conductivity, photoluminescence, and chemical inertness. Here, we investigated different methodologies for the exfoliation of BN nanosheets (BNNs), using ball milling and ultrasound processing, the latter using both an ultrasound bath and tip sonication. The best results are obtained using tip sonication, which leads to the formation of few-layered nanosheets with a narrow size distribution. Importantly, it was observed that with the addition of pluronic acid F127 to the medium, there was a significant improvement in the BN nanosheets (BNNs) production yield. Moreover, the resultant BNNs present improved stability in an aqueous solution. Cytotoxicity studies performed with HeLa cells showed the importance of taking into account the possible interferences of the nanomaterial with the selected assay. The prepared BNNs coated with pluronic presented improved cytotoxicity at concentrations up to 200 \u03bcg mL\u22121 with more than 90% viability after 24 h of incubation. Confocal microscopy also showed high cell internalization of the nanomaterials and their preferential biodistribution in the cell cytoplasm.<\/jats:p>","DOI":"10.3390\/biomedicines10123238","type":"journal-article","created":{"date-parts":[[2022,12,13]],"date-time":"2022-12-13T04:32:48Z","timestamp":1670905968000},"page":"3238","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Sustainable Synthesis of Highly Biocompatible 2D Boron Nitride Nanosheets"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1048-8869","authenticated-orcid":false,"given":"Marina","family":"Llenas","sequence":"first","affiliation":[{"name":"Institut de Ci\u00e8ncia de Materials de Barcelona (ICMAB-CSIC), Campus de la UAB, 08193 Bellaterra, Spain"}]},{"given":"Lorenzo","family":"Cuenca","sequence":"additional","affiliation":[{"name":"Institut de Ci\u00e8ncia de Materials de Barcelona (ICMAB-CSIC), Campus de la UAB, 08193 Bellaterra, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6315-7433","authenticated-orcid":false,"given":"Carla","family":"Santos","sequence":"additional","affiliation":[{"name":"LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal"},{"name":"CQE\u2014Centro de Qu\u00edmica Estrutural, Instituto Superior T\u00e9cnico, University of Lisbon, 1049-001 Lisbon, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6318-1425","authenticated-orcid":false,"given":"Igor","family":"Bdikin","sequence":"additional","affiliation":[{"name":"TEMA-Nanotechnology Research Group, Mechanical Engineering Department, University of Aveiro, 3810-193 Aveiro, Portugal"},{"name":"Intelligent Systems Associate Laboratory (LASI), 3810-193 Aveiro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2302-9661","authenticated-orcid":false,"given":"Gil","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"TEMA-Nanotechnology Research Group, Mechanical Engineering Department, University of Aveiro, 3810-193 Aveiro, Portugal"},{"name":"Intelligent Systems Associate Laboratory (LASI), 3810-193 Aveiro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7116-2152","authenticated-orcid":false,"given":"Gerard","family":"Tob\u00edas-Rossell","sequence":"additional","affiliation":[{"name":"Institut de Ci\u00e8ncia de Materials de Barcelona (ICMAB-CSIC), Campus de la UAB, 08193 Bellaterra, Spain"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6409","DOI":"10.1021\/acs.chemrev.7b00727","article-title":"Two-Dimensional Metal Nanomaterials: Synthesis, Properties, and Applications","volume":"118","author":"Chen","year":"2018","journal-title":"Chem. 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