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Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3

Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone density over time. It affects one in two women and one in four men, age 50 and older. New treatments that specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, that acts downstre...

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Detalles Bibliográficos
Autores principales: Vrathasha, Vrathasha, Booksh, Karl, Duncan, Randall L., Nohe, Anja
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071089/
https://www.ncbi.nlm.nih.gov/pubmed/29987256
http://dx.doi.org/10.3390/nano8070513
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author Vrathasha, Vrathasha
Booksh, Karl
Duncan, Randall L.
Nohe, Anja
author_facet Vrathasha, Vrathasha
Booksh, Karl
Duncan, Randall L.
Nohe, Anja
author_sort Vrathasha, Vrathasha
collection PubMed
description Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone density over time. It affects one in two women and one in four men, age 50 and older. New treatments that specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, that acts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesis in-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cells remains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3 to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by size exclusion chromatography and verified the conjugation and stability using UV/VIS and Fourier transform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s and the conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biological response similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealed that CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacological inhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized by caveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cell through caveolae mediated endocytosis.
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spelling pubmed-60710892018-08-09 Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3 Vrathasha, Vrathasha Booksh, Karl Duncan, Randall L. Nohe, Anja Nanomaterials (Basel) Article Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone density over time. It affects one in two women and one in four men, age 50 and older. New treatments that specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, that acts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesis in-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cells remains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3 to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by size exclusion chromatography and verified the conjugation and stability using UV/VIS and Fourier transform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s and the conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biological response similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealed that CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacological inhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized by caveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cell through caveolae mediated endocytosis. MDPI 2018-07-09 /pmc/articles/PMC6071089/ /pubmed/29987256 http://dx.doi.org/10.3390/nano8070513 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Vrathasha, Vrathasha
Booksh, Karl
Duncan, Randall L.
Nohe, Anja
Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
title Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
title_full Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
title_fullStr Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
title_full_unstemmed Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
title_short Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
title_sort mechanisms of cellular internalization of quantum dot® conjugated bone formation mimetic peptide ck2.3
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071089/
https://www.ncbi.nlm.nih.gov/pubmed/29987256
http://dx.doi.org/10.3390/nano8070513
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