Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9

Elaheh Sadat Hosseini,1 Maryam Nikkhah,1 Saman Hosseinkhani21Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; 2Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IranPurpose: Gene therapy has becom...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Hosseini ES, Nikkhah M, Hosseinkhani S
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2019
Materias:	Cationic liposomes Cholesterol domains PEGylation Cas9/sgRNA delivery Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/c74c16c3b99c439e8523af1bf9f2b9d6
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:c74c16c3b99c439e8523af1bf9f2b9d6
record_format	dspace
spelling	oai:doaj.org-article:c74c16c3b99c439e8523af1bf9f2b9d62021-12-02T10:55:30ZCholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas91178-2013https://doaj.org/article/c74c16c3b99c439e8523af1bf9f2b9d62019-06-01T00:00:00Zhttps://www.dovepress.com/cholesterol-rich-lipid-mediated-nanoparticles-boost-of-transfection-ef-peer-reviewed-article-IJNhttps://doaj.org/toc/1178-2013Elaheh Sadat Hosseini,1 Maryam Nikkhah,1 Saman Hosseinkhani21Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; 2Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IranPurpose: Gene therapy has become a promising remedy to treat disease by modifying the person’s genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability.Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum.Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.Keywords: cationic liposomes, cholesterol domains, PEGylation, Cas9/sgRNA deliveryHosseini ESNikkhah MHosseinkhani SDove Medical PressarticleCationic liposomesCholesterol domainsPEGylationCas9/sgRNA deliveryMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol Volume 14, Pp 4353-4366 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cationic liposomes Cholesterol domains PEGylation Cas9/sgRNA delivery Medicine (General) R5-920
spellingShingle	Cationic liposomes Cholesterol domains PEGylation Cas9/sgRNA delivery Medicine (General) R5-920 Hosseini ES Nikkhah M Hosseinkhani S Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
description	Elaheh Sadat Hosseini,1 Maryam Nikkhah,1 Saman Hosseinkhani21Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; 2Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IranPurpose: Gene therapy has become a promising remedy to treat disease by modifying the person’s genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability.Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum.Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.Keywords: cationic liposomes, cholesterol domains, PEGylation, Cas9/sgRNA delivery
format	article
author	Hosseini ES Nikkhah M Hosseinkhani S
author_facet	Hosseini ES Nikkhah M Hosseinkhani S
author_sort	Hosseini ES
title	Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
title_short	Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
title_full	Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
title_fullStr	Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
title_full_unstemmed	Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
title_sort	cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by crispr-cas9
publisher	Dove Medical Press
publishDate	2019
url	https://doaj.org/article/c74c16c3b99c439e8523af1bf9f2b9d6
work_keys_str_mv	AT hosseinies cholesterolrichlipidmediatednanoparticlesboostoftransfectionefficiencyutilizedforgeneeditingbycrisprcas9 AT nikkhahm cholesterolrichlipidmediatednanoparticlesboostoftransfectionefficiencyutilizedforgeneeditingbycrisprcas9 AT hosseinkhanis cholesterolrichlipidmediatednanoparticlesboostoftransfectionefficiencyutilizedforgeneeditingbycrisprcas9
_version_	1718396493529874432

Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9

Ejemplares similares