Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro
To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fi...
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description | To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro.
First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.
The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.
All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery. |
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First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.
The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.
All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.</description><identifier>EISSN: 1090-0535</identifier><identifier>PMID: 19137061</identifier><language>eng</language><publisher>United States</publisher><subject>Analysis of Variance ; Cell Proliferation ; Cells, Cultured ; Chitosan - chemistry ; Connective Tissue Cells - cytology ; Connective Tissue Cells - metabolism ; Down-Regulation ; Eye - cytology ; Fibroblasts - cytology ; Fibroblasts - metabolism ; HeLa Cells ; Humans ; I-kappa B Kinase - genetics ; I-kappa B Kinase - metabolism ; NF-kappa B - metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Particle Size ; Polyethylene Glycols - chemistry ; Polyethyleneimine - chemistry ; Polymers - chemical synthesis ; Polymers - chemistry ; Polymers - metabolism ; RNA Interference ; RNA, Small Interfering - genetics ; RNA, Small Interfering - metabolism ; Transfection</subject><ispartof>Molecular vision, 2008, Vol.14, p.2616-2628</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,4024</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19137061$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Duan, Yongheng</creatorcontrib><creatorcontrib>Guan, Xipeng</creatorcontrib><creatorcontrib>Ge, Jian</creatorcontrib><creatorcontrib>Quan, Daping</creatorcontrib><creatorcontrib>Zhuo, Yehong</creatorcontrib><creatorcontrib>Ye, Hehua</creatorcontrib><creatorcontrib>Shao, Tingting</creatorcontrib><title>Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro</title><title>Molecular vision</title><addtitle>Mol Vis</addtitle><description>To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro.
First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.
The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.
All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.</description><subject>Analysis of Variance</subject><subject>Cell Proliferation</subject><subject>Cells, Cultured</subject><subject>Chitosan - chemistry</subject><subject>Connective Tissue Cells - cytology</subject><subject>Connective Tissue Cells - metabolism</subject><subject>Down-Regulation</subject><subject>Eye - cytology</subject><subject>Fibroblasts - cytology</subject><subject>Fibroblasts - metabolism</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>I-kappa B Kinase - genetics</subject><subject>I-kappa B Kinase - metabolism</subject><subject>NF-kappa B - metabolism</subject><subject>Nuclear Magnetic Resonance, Biomolecular</subject><subject>Particle Size</subject><subject>Polyethylene Glycols - chemistry</subject><subject>Polyethyleneimine - chemistry</subject><subject>Polymers - chemical synthesis</subject><subject>Polymers - chemistry</subject><subject>Polymers - metabolism</subject><subject>RNA Interference</subject><subject>RNA, Small Interfering - genetics</subject><subject>RNA, Small Interfering - metabolism</subject><subject>Transfection</subject><issn>1090-0535</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo1kE1LAzEYhBdBbK3-BclJTwvJfqSbYyl-lBYF6X15k33TRrLJmmSFHv3nLlpPA8MzwzAX2ZxRQXNal_Usu47xg9KC1dXyKpsxwcol5Wyefa8hGe-MIg6cz5UfvD31GCLpsTOQsCOb7VZiApIgHDAZdyDRvL-uiHFHI00i6YhkCN4ajeG3jHhNjmMPjuzRefcQiYIhjhaJNjJ4aSGmOMXJl0nB32SXGmzE27Musv3T4379ku_enjfr1S4f6orlCMsCmaJ1ozkVspo8yZtOKSZ4g1BK2tWcd4JJVRRUl1XDS8kYE6ABeafKRXb_VztN_RwxprY3UaG14NCPseVCVBPfTODdGRzl9EE7BNNDOLX_n5U_82xpiA</recordid><startdate>2008</startdate><enddate>2008</enddate><creator>Duan, Yongheng</creator><creator>Guan, Xipeng</creator><creator>Ge, Jian</creator><creator>Quan, Daping</creator><creator>Zhuo, Yehong</creator><creator>Ye, Hehua</creator><creator>Shao, Tingting</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>2008</creationdate><title>Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro</title><author>Duan, Yongheng ; Guan, Xipeng ; Ge, Jian ; Quan, Daping ; Zhuo, Yehong ; Ye, Hehua ; Shao, Tingting</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p541-ea72e1c058f609b4541b68dcc1968ea3b0d566d91bc220f34863b1119afae6dc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Analysis of Variance</topic><topic>Cell Proliferation</topic><topic>Cells, Cultured</topic><topic>Chitosan - chemistry</topic><topic>Connective Tissue Cells - cytology</topic><topic>Connective Tissue Cells - metabolism</topic><topic>Down-Regulation</topic><topic>Eye - cytology</topic><topic>Fibroblasts - cytology</topic><topic>Fibroblasts - metabolism</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>I-kappa B Kinase - genetics</topic><topic>I-kappa B Kinase - metabolism</topic><topic>NF-kappa B - metabolism</topic><topic>Nuclear Magnetic Resonance, Biomolecular</topic><topic>Particle Size</topic><topic>Polyethylene Glycols - chemistry</topic><topic>Polyethyleneimine - chemistry</topic><topic>Polymers - chemical synthesis</topic><topic>Polymers - chemistry</topic><topic>Polymers - metabolism</topic><topic>RNA Interference</topic><topic>RNA, Small Interfering - genetics</topic><topic>RNA, Small Interfering - metabolism</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Duan, Yongheng</creatorcontrib><creatorcontrib>Guan, Xipeng</creatorcontrib><creatorcontrib>Ge, Jian</creatorcontrib><creatorcontrib>Quan, Daping</creatorcontrib><creatorcontrib>Zhuo, Yehong</creatorcontrib><creatorcontrib>Ye, Hehua</creatorcontrib><creatorcontrib>Shao, Tingting</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular vision</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Duan, Yongheng</au><au>Guan, Xipeng</au><au>Ge, Jian</au><au>Quan, Daping</au><au>Zhuo, Yehong</au><au>Ye, Hehua</au><au>Shao, Tingting</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro</atitle><jtitle>Molecular vision</jtitle><addtitle>Mol Vis</addtitle><date>2008</date><risdate>2008</risdate><volume>14</volume><spage>2616</spage><epage>2628</epage><pages>2616-2628</pages><eissn>1090-0535</eissn><abstract>To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro.
First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.
The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.
All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.</abstract><cop>United States</cop><pmid>19137061</pmid><tpages>13</tpages></addata></record> |
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subjects | Analysis of Variance Cell Proliferation Cells, Cultured Chitosan - chemistry Connective Tissue Cells - cytology Connective Tissue Cells - metabolism Down-Regulation Eye - cytology Fibroblasts - cytology Fibroblasts - metabolism HeLa Cells Humans I-kappa B Kinase - genetics I-kappa B Kinase - metabolism NF-kappa B - metabolism Nuclear Magnetic Resonance, Biomolecular Particle Size Polyethylene Glycols - chemistry Polyethyleneimine - chemistry Polymers - chemical synthesis Polymers - chemistry Polymers - metabolism RNA Interference RNA, Small Interfering - genetics RNA, Small Interfering - metabolism Transfection |
title | Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro |
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