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Lyophilization Based Isolation of Exosomes

Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophil...

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Autores principales: Qazi, Rida e Maria, Sajid, Zahra, Zhao, Chunqiu, Hussain, Irfan, Iftikhar, Fizza, Jameel, Muhammad, Rehman, Fawad Ur, Mian, Afsar Ali
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341925/
https://www.ncbi.nlm.nih.gov/pubmed/37445655
http://dx.doi.org/10.3390/ijms241310477
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author Qazi, Rida e Maria
Sajid, Zahra
Zhao, Chunqiu
Hussain, Irfan
Iftikhar, Fizza
Jameel, Muhammad
Rehman, Fawad Ur
Mian, Afsar Ali
author_facet Qazi, Rida e Maria
Sajid, Zahra
Zhao, Chunqiu
Hussain, Irfan
Iftikhar, Fizza
Jameel, Muhammad
Rehman, Fawad Ur
Mian, Afsar Ali
author_sort Qazi, Rida e Maria
collection PubMed
description Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.
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spelling pubmed-103419252023-07-14 Lyophilization Based Isolation of Exosomes Qazi, Rida e Maria Sajid, Zahra Zhao, Chunqiu Hussain, Irfan Iftikhar, Fizza Jameel, Muhammad Rehman, Fawad Ur Mian, Afsar Ali Int J Mol Sci Article Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells. MDPI 2023-06-22 /pmc/articles/PMC10341925/ /pubmed/37445655 http://dx.doi.org/10.3390/ijms241310477 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Qazi, Rida e Maria
Sajid, Zahra
Zhao, Chunqiu
Hussain, Irfan
Iftikhar, Fizza
Jameel, Muhammad
Rehman, Fawad Ur
Mian, Afsar Ali
Lyophilization Based Isolation of Exosomes
title Lyophilization Based Isolation of Exosomes
title_full Lyophilization Based Isolation of Exosomes
title_fullStr Lyophilization Based Isolation of Exosomes
title_full_unstemmed Lyophilization Based Isolation of Exosomes
title_short Lyophilization Based Isolation of Exosomes
title_sort lyophilization based isolation of exosomes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341925/
https://www.ncbi.nlm.nih.gov/pubmed/37445655
http://dx.doi.org/10.3390/ijms241310477
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