Optimized Solid‐Phase‐Assisted Synthesis of Oleic Acid Containing siRNA Nanocarriers

Reinhard, Sören; Zhang, Wei; Wagner, Ernst

doi:10.1002/cmdc.201700350

Cited by 23 publications

(31 citation statements)

References 67 publications

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“…In case of oligomers 440 , 835, and 454 a cleavage cocktail TFA/EDT/H 2 O/TIS 94:2.5:2.5:1 (v/v) at a ratio of 10 mL g −1 resin and in case of post‐PEGylation agents TFA/H 2 O/TIS 95:2.5:2.5 was chosen. Cleavage was conducted for 90 min with the exception of 454 , here the cleavage solution was precooled to 4 °C and cleavage was only performed for 30 min to prevent side reaction at the double bond of the oleic acid . The cleaved oligomers were precipitated in a 50:50 (v/v) mixture of n‐hexane/MTBE and purified by size exclusion chromatography (SEC) performed with 10 × 10 −3 m hydrochloric acid/acetonitrile 7:3 as solvent.…”

Section: Methodsmentioning

confidence: 99%

“…Cleavage was conducted for 90 min with the exception of 454, here the cleavage solution was precooled to 4 °C and cleavage was only performed for 30 min to prevent side reaction at the double bond of the oleic acid. [69] The cleaved oligomers were precipitated in a 50:50 (v/v) mixture of n-hexane/MTBE and purified by size exclusion chromatography (SEC) performed with 10 × 10 −3 m hydrochloric acid/acetonitrile 7:3 as solvent. An ÄKTA purifier system (GE Healthcare Biosciences, Uppsala, Sweden) equipped with a Sephadex G-10 column and a P-900 solvent pump module, a UV-900 spectrophotometrical detector, a pH/C-900 conductivity module and a Frac-950 automated fractionator were used.…”

Section: Peptide Synthesismentioning

confidence: 99%

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EGFR Targeting and Shielding of pDNA Lipopolyplexes via Bivalent Attachment of a Sequence‐Defined PEG Agent

Morys

Urnauer

Spitzweg

et al. 2017

Macromolecular Bioscience

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For successful nonviral gene delivery, cationic polymers are promising DNA carrier, which need to comprise several functionalities. The current work focuses on the postincorporation of epidermal growth factor receptor (EGFR) targeted PEGylation agents onto lipopolyplexes for pDNA delivery. T‐shaped lipo‐oligomers are previously found to be effective sequence‐defined carriers for pDNA and siRNA. Here, the bis‐oleoyl‐oligoaminoethanamide 454 containing tyrosine trimer–cysteine ends is applied for complex formation with pDNA coding for luciferase or sodium iodide symporter (NIS). In a second step, the lipopolyplexes are modified via disulfide formation with sequence‐defined monovalent or bivalent PEGylation agents containing one or two 3‐nitro‐2‐pyridinesulfenyl (NPys)‐activated cysteines, respectively. For targeting, the polyethylene glycol (PEG) agents comprise the EGFR targeting peptide GE11. In comparison of all transfection complexes, 454 lipopolyplexes modified with the bidentate PEG–GE11 agent show the best, EGFR‐dependent uptake as well as luciferase and NIS gene expression into receptor‐positive tumor cells.

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Section: Methodsmentioning

confidence: 99%

Section: Peptide Synthesismentioning

confidence: 99%

EGFR Targeting and Shielding of pDNA Lipopolyplexes via Bivalent Attachment of a Sequence‐Defined PEG Agent

Morys

Urnauer

Spitzweg

et al. 2017

Macromolecular Bioscience

Self Cite

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“…Different fatty acids in oligo-amidoamines have been shown to induce membrane leakage in erythrocytes and to increase cell death in in vitro cell assays (Klein et al, 2016;Reinhard, Zhang & Wagner, 2017). Influence of target formulations completely prepared with microfluidics on metabolic activity of KB cells was assessed by MTT assay to account for any apparent effects on cell survivability.…”

Section: Toxicitymentioning

confidence: 99%

A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

Loy

Klein

Krzysztoń

et al. 2019

PeerJ Materials Science

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Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid's transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed "oligomers") with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding polyethylene glycol (PEG) and cell targeting folic acid (termed "PEG-ligands") as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azidealkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core-lipid anchor-PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to n core oligomer ) showed optimal siRNA mediated gene knock-down when containing defined PEG domains of in sum 24 and 36 ethylene oxide repetitions, 12 EOs each from the lipid anchor and 12 or 24 EOs from the PEG-ligand, respectively. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core-PEG-ligand polyplexes formed by click modification of How to cite this article Loy DM, Klein PM, Krzysztoń R, Lächelt U, Rädler JO, Wagner E. 2019. A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship.

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“…Analytical, Inorganic, Organic, Physical, Materials Science Toxicity Core (CO + siRNA) -lipid anchor -PEG-ligand polyplexes do not alter the metabolic activity profile of KB cells in comparison to core polyplexes alone. Different fatty acids in oligo-amidoamines have been shown to induce membrane leakage in erythrocytes and to increase cell death in in vitro cell assays (Klein et al, 2016;Reinhard, Zhang, & Wagner, 2017). Influence of target formulations completely prepared with microfluidics on metabolic activity of KB cells was assessed by MTT assay to account for any apparent effects on cell survivability.…”

Section: Chemistry Journalsmentioning

confidence: 99%

Peer Review #1 of "A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship (v0.2)"

2019

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Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid's transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed 'oligomers') with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding PEG and cell targeting folic acid (termed 'PEG-ligands') as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide-alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core-lipid anchor-PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEGligands (relative to n core oligomer) showed optimal siRNA mediated gene knock-down when

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Optimized Solid‐Phase‐Assisted Synthesis of Oleic Acid Containing siRNA Nanocarriers

Cited by 23 publications

References 67 publications

EGFR Targeting and Shielding of pDNA Lipopolyplexes via Bivalent Attachment of a Sequence‐Defined PEG Agent

EGFR Targeting and Shielding of pDNA Lipopolyplexes via Bivalent Attachment of a Sequence‐Defined PEG Agent

A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

Peer Review #1 of "A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship (v0.2)"

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