Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles
Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the...
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sg-ntu-dr.10356-1645782023-07-14T16:07:38Z Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles Bonany, Mar Pérez-Berná, Ana Joaquina Dučić, Tanja Pereiro, Eva Martin-Gómez, Helena Mas-Moruno, Carlos van Rijt, Sabine Zhao, Zhitong Espanol, Montserrat Ginebra, Maria-Pau School of Materials Science and Engineering Engineering::Materials Hydroxyapatite Nanoparticles Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form. Published version The authors acknowledge the Spanish Government for financial support through the PID2019-103892RB-I00/AEI/10.13039/501100011033 and MAT2017-83905-R projects, the “Ayudas para contratos predoctorales para la formaci ́on de doctores” scholarship (BES-2016-07672) of M. Bonany and a Ramon y Cajal grant of C. Mas-Moruno. They also thank the Generalitat de Catalunya for funding through project 2017SGR-1165, the Serra Hunter Fellowship of M.Espanol and the ICREA Academia Award of M-P. Ginebra. 2023-02-06T00:47:57Z 2023-02-06T00:47:57Z 2022 Journal Article Bonany, M., Pérez-Berná, A. J., Dučić, T., Pereiro, E., Martin-Gómez, H., Mas-Moruno, C., van Rijt, S., Zhao, Z., Espanol, M. & Ginebra, M. (2022). Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles. Biomaterials Advances, 142, 213148-. https://dx.doi.org/10.1016/j.bioadv.2022.213148 2772-9508 https://hdl.handle.net/10356/164578 10.1016/j.bioadv.2022.213148 36274359 2-s2.0-85140266416 142 213148 en Biomaterials Advances © 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). application/pdf |
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Engineering::Materials Hydroxyapatite Nanoparticles Bonany, Mar Pérez-Berná, Ana Joaquina Dučić, Tanja Pereiro, Eva Martin-Gómez, Helena Mas-Moruno, Carlos van Rijt, Sabine Zhao, Zhitong Espanol, Montserrat Ginebra, Maria-Pau Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
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Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form. |
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School of Materials Science and Engineering |
author_facet |
School of Materials Science and Engineering Bonany, Mar Pérez-Berná, Ana Joaquina Dučić, Tanja Pereiro, Eva Martin-Gómez, Helena Mas-Moruno, Carlos van Rijt, Sabine Zhao, Zhitong Espanol, Montserrat Ginebra, Maria-Pau |
format |
Article |
author |
Bonany, Mar Pérez-Berná, Ana Joaquina Dučić, Tanja Pereiro, Eva Martin-Gómez, Helena Mas-Moruno, Carlos van Rijt, Sabine Zhao, Zhitong Espanol, Montserrat Ginebra, Maria-Pau |
author_sort |
Bonany, Mar |
title |
Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
title_short |
Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
title_full |
Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
title_fullStr |
Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
title_full_unstemmed |
Hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
title_sort |
hydroxyapatite nanoparticles-cell interaction: new approaches to disclose the fate of membrane-bound and internalised nanoparticles |
publishDate |
2023 |
url |
https://hdl.handle.net/10356/164578 |
_version_ |
1773551421021487104 |