Combinatorial Synthesis of a Lipidoid Library by Thiolactone Chemistry: In Vitro Screening and In Vivo Validation for siRNA Delivery

Abstract

Transcriptional inhibition by small interfering RNA (siRNA) delivery using synthetic transfection agents eliminates the subsequent risk of introducing mutations in relevant genes, as opposed to viral vectors. However, synthetic vectors with comparable transfection efficiency to that of viral vectors are yet to be developed. Hence, synthesizing new transfection vehicles with low toxicity is important. In this study, a library of lipid-like molecules (lipidoids) was synthesized by thiolactone chemistry. This library facilitated nonviral delivery of siRNA to mammalian cells, inducing sequence-specific knockdown of a target gene. The liposomal nanoparticles complexed with anti-green fluorescent protein (GFP) siRNA were successfully screened for transfection efficiency using a HeLa-GFP cell line. The five best-performing lipidoids identified in the screening were found to exhibit superior GFP-knockdown efficiency compared with commercially available transfection reagents. The efficiency of siRNA delivery by one of these lipidoids with minimal toxicity was further successfully evaluated in vivo using Kdrl:EGFP zebrafish embryos as a model system. Our study would be important as a facile synthetic route of efficient nonviral nucleic acid delivery to live cells and organisms.

WRAP-based nanoparticles for siRNA delivery in zebrafish embryos by simple bath immersion

“…In this study, we established a protocol for efficient and specific gene silencing by immersing zebrafish embryos in an E3 medium containing PEGylated WRAP1-based nanoparticles encapsulating siRNA. First, we confirmed the silencing efficiency of three different published siRNAs targeting the GFP protein 17 , 18 , 19 in GFP-overexpressing HEK293 cells. Transfections were performed with the WRAP-based nanoparticles as previously described, 13 , 14 , 15 and the GFP silencing of three different siGFP-loaded nanoparticles in GFP-overexpressing HEK293 cells was evaluated by western blot in comparison to the siRNA transfection without delivery system ( Figure 2 ).…”

“…Therefore, many different siRNA sequences have been published in the literature or even proposed by various manufacturers. For this study, we selected three different siRNAs (siGFP-M, 17 siGFP-G, 18 and siGFP-W 19 , with the letter corresponding to the name of the first author) to evaluate their silencing properties using cellular delivery systems of the WRAP peptides we developed. 13 Indeed, the use of different siRNAs may lead to the induction of different silencing activities, depending on the model employed (i.e., cellular or animal).…”

WRAP-based nanoparticles for siRNA delivery in zebrafish embryos by simple bath immersion

“…In this study, we established a protocol for efficient and specific gene silencing by immersing zebrafish embryos in an E3 medium containing PEGylated WRAP1-based nanoparticles encapsulating siRNA. First, we confirmed the silencing efficiency of three different published siRNAs targeting the GFP protein 17 , 18 , 19 in GFP-overexpressing HEK293 cells. Transfections were performed with the WRAP-based nanoparticles as previously described, 13 , 14 , 15 and the GFP silencing of three different siGFP-loaded nanoparticles in GFP-overexpressing HEK293 cells was evaluated by western blot in comparison to the siRNA transfection without delivery system ( Figure 2 ).…”

“…Therefore, many different siRNA sequences have been published in the literature or even proposed by various manufacturers. For this study, we selected three different siRNAs (siGFP-M, 17 siGFP-G, 18 and siGFP-W 19 , with the letter corresponding to the name of the first author) to evaluate their silencing properties using cellular delivery systems of the WRAP peptides we developed. 13 Indeed, the use of different siRNAs may lead to the induction of different silencing activities, depending on the model employed (i.e., cellular or animal).…”

Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects

“…Those materials were formulated as liposomes to deliver siRNA and tested on HeLa-GFP cells. Five of the formulations showed superior knock-down efficiency than the commercial reagents, emphasizing the importance of lipidoid structure for transfection capability as well as the liposome formulation attributes such as particle size ( 39 ). These findings may pave a foundation for further exploration of lipidoids for gene delivery in the future.…”

Lipids and Lipid Derivatives for RNA Delivery

“…Kdrl:EGFP Zebrafish embryos injected with T18U-PY18-A4 LNPs encapsulating anti-GFP siRNA showed a significant reduction in EGFP expression in blood vessels. 602 In 2019, Miao et al developed a library of 1080 ionizable lipids 603 via isocyanide-mediated three-component reaction (3-CR) 657 that simultaneously coupled primary or secondary amines, 658 ketones with different alkyl tail lengths and different degrees of saturation, 648,659 and isocyanides or isocyanide derivatives (Figure 68). 660 Results showed that lipids with longer, unsaturated tails showed higher mRNA delivery efficiency, and A2-Iso5-2DC18 (A2) and A12-Iso5−2DC18 (A12) were the top-performing lipids for mRNA delivery in bone marrow-derived dendritic cells (BMDCs), bone marrow- derived macrophages (BMDMs), and Hela cells.…”

“…The combination of this method with high-throughput screening can lead to the quick discovery of ionizable lipids with good RNA delivery efficiency. ,− Many types of reactions can be applied to produce lipidoids, including aza-Michael addition of amines with acrylamides or acrylesters, ,, ring-opening of epoxides with amines, ,, and reductive amination of aldehydes − (Figure ). Thiol–yne click reaction , and multicomponent reactions − can also be used in the synthesis of lipidoids (Figure ).…”

WRAP-based nanoparticles for siRNA delivery in zebrafish embryos by simple bath immersion

“…In this study, we established a protocol for efficient and specific gene silencing by immersing zebrafish embryos in an E3 medium containing PEGylated WRAP1-based nanoparticles encapsulating siRNA. First, we confirmed the silencing efficiency of three different published siRNAs targeting the GFP protein 17 , 18 , 19 in GFP-overexpressing HEK293 cells. Transfections were performed with the WRAP-based nanoparticles as previously described, 13 , 14 , 15 and the GFP silencing of three different siGFP-loaded nanoparticles in GFP-overexpressing HEK293 cells was evaluated by western blot in comparison to the siRNA transfection without delivery system ( Figure 2 ).…”

“…Therefore, many different siRNA sequences have been published in the literature or even proposed by various manufacturers. For this study, we selected three different siRNAs (siGFP-M, 17 siGFP-G, 18 and siGFP-W 19 , with the letter corresponding to the name of the first author) to evaluate their silencing properties using cellular delivery systems of the WRAP peptides we developed. 13 Indeed, the use of different siRNAs may lead to the induction of different silencing activities, depending on the model employed (i.e., cellular or animal).…”

Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects

“…Those materials were formulated as liposomes to deliver siRNA and tested on HeLa-GFP cells. Five of the formulations showed superior knock-down efficiency than the commercial reagents, emphasizing the importance of lipidoid structure for transfection capability as well as the liposome formulation attributes such as particle size ( 39 ). These findings may pave a foundation for further exploration of lipidoids for gene delivery in the future.…”

Lipids and Lipid Derivatives for RNA Delivery

“…Kdrl:EGFP Zebrafish embryos injected with T18U-PY18-A4 LNPs encapsulating anti-GFP siRNA showed a significant reduction in EGFP expression in blood vessels. 602 In 2019, Miao et al developed a library of 1080 ionizable lipids 603 via isocyanide-mediated three-component reaction (3-CR) 657 that simultaneously coupled primary or secondary amines, 658 ketones with different alkyl tail lengths and different degrees of saturation, 648,659 and isocyanides or isocyanide derivatives (Figure 68). 660 Results showed that lipids with longer, unsaturated tails showed higher mRNA delivery efficiency, and A2-Iso5-2DC18 (A2) and A12-Iso5−2DC18 (A12) were the top-performing lipids for mRNA delivery in bone marrow-derived dendritic cells (BMDCs), bone marrow- derived macrophages (BMDMs), and Hela cells.…”

“…The combination of this method with high-throughput screening can lead to the quick discovery of ionizable lipids with good RNA delivery efficiency. ,− Many types of reactions can be applied to produce lipidoids, including aza-Michael addition of amines with acrylamides or acrylesters, ,, ring-opening of epoxides with amines, ,, and reductive amination of aldehydes − (Figure ). Thiol–yne click reaction , and multicomponent reactions − can also be used in the synthesis of lipidoids (Figure ).…”

WRAP-based nanoparticles for siRNA delivery in zebrafish embryos by simple bath immersion

“…In this study, we established a protocol for efficient and specific gene silencing by immersing zebrafish embryos in an E3 medium containing PEGylated WRAP1-based nanoparticles encapsulating siRNA. First, we confirmed the silencing efficiency of three different published siRNAs targeting the GFP protein 17 , 18 , 19 in GFP-overexpressing HEK293 cells. Transfections were performed with the WRAP-based nanoparticles as previously described, 13 , 14 , 15 and the GFP silencing of three different siGFP-loaded nanoparticles in GFP-overexpressing HEK293 cells was evaluated by western blot in comparison to the siRNA transfection without delivery system ( Figure 2 ).…”

“…Therefore, many different siRNA sequences have been published in the literature or even proposed by various manufacturers. For this study, we selected three different siRNAs (siGFP-M, 17 siGFP-G, 18 and siGFP-W 19 , with the letter corresponding to the name of the first author) to evaluate their silencing properties using cellular delivery systems of the WRAP peptides we developed. 13 Indeed, the use of different siRNAs may lead to the induction of different silencing activities, depending on the model employed (i.e., cellular or animal).…”

Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects

“…Those materials were formulated as liposomes to deliver siRNA and tested on HeLa-GFP cells. Five of the formulations showed superior knock-down efficiency than the commercial reagents, emphasizing the importance of lipidoid structure for transfection capability as well as the liposome formulation attributes such as particle size ( 39 ). These findings may pave a foundation for further exploration of lipidoids for gene delivery in the future.…”

Lipids and Lipid Derivatives for RNA Delivery

“…Kdrl:EGFP Zebrafish embryos injected with T18U-PY18-A4 LNPs encapsulating anti-GFP siRNA showed a significant reduction in EGFP expression in blood vessels. 602 In 2019, Miao et al developed a library of 1080 ionizable lipids 603 via isocyanide-mediated three-component reaction (3-CR) 657 that simultaneously coupled primary or secondary amines, 658 ketones with different alkyl tail lengths and different degrees of saturation, 648,659 and isocyanides or isocyanide derivatives (Figure 68). 660 Results showed that lipids with longer, unsaturated tails showed higher mRNA delivery efficiency, and A2-Iso5-2DC18 (A2) and A12-Iso5−2DC18 (A12) were the top-performing lipids for mRNA delivery in bone marrow-derived dendritic cells (BMDCs), bone marrow- derived macrophages (BMDMs), and Hela cells.…”

“…The combination of this method with high-throughput screening can lead to the quick discovery of ionizable lipids with good RNA delivery efficiency. ,− Many types of reactions can be applied to produce lipidoids, including aza-Michael addition of amines with acrylamides or acrylesters, ,, ring-opening of epoxides with amines, ,, and reductive amination of aldehydes − (Figure ). Thiol–yne click reaction , and multicomponent reactions − can also be used in the synthesis of lipidoids (Figure ).…”