Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles

Krzysztoń, Rafał; Salem, B.; Lee, Dian-Jang; Schwake, Gerlinde; Wagner, Ernst; Rädler, Joachim O.

doi:10.1039/c7nr01593c

Cited by 60 publications

(43 citation statements)

References 44 publications

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“…The resulting liposomes were large unilamellar vesicles or small multilamellar vesicles depending on the antisense oligonucleotide‐to‐lipid ratio used . More recently, greater control was achieved over the mixing process when performed by T‐junction mixing, microfluidic mixing using an SHM, or microfluidic hydrodynamic focusing (MHF) (see Section ). Depending on the lipid formulation, nucleic acid payload, and production method, particles containing an electron‐dense core were produced for T‐junction mixing and SHM with reported encapsulation efficiencies of >90% .…”

Section: Design Principles For Lipid Nanoparticles For Sirna Deliverymentioning

confidence: 99%

State‐of‐the‐Art Design and Rapid‐Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

et al. 2018

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The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smtd.201700375.Lipid nanoparticles (LNPs) are currently the most clinically advanced nonviral carriers for the delivery of small interfering RNA (siRNA). Free siRNA molecules suffer from unfavorable physicochemical characteristics and rapid clearance mechanisms, hampering the ability to reach the cytoplasm of target cells when administered intravenously. As a result, the therapeutic use of siRNA is crucially dependent on delivery strategies. LNPs can encapsulate siRNA to protect it from degradative endonucleases in the circulation, prevent kidney clearance, and provide a vehicle to deliver siRNA in the cell and induce its subsequent release into the cytoplasm. Here, the structure and composition of LNP-siRNA are described including how these affect their pharmacokinetic parameters and gene-silencing activity. In addition, the evolution of LNP-siRNA production methods is discussed, as the development of rapid-mixing platforms for the reproducible and scalable manufacturing has facilitated entry of LNP-siRNA into the clinic over the last decade. Finally, the potential of LNPs in delivering other nucleic acids, such as messenger RNA and CRISPR/Cas9 components, is highlighted alongside how a design-of-experiment approach may be used to improve the efficacy of LNP formulations. Nucleic Acid Delivery

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Section: Design Principles For Lipid Nanoparticles For Sirna Deliverymentioning

confidence: 99%

State‐of‐the‐Art Design and Rapid‐Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

et al. 2018

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“…Continuous or batch wise production of PEI pDNA polyplexes with active mixing by surface acoustic waves (SAWs) has been demonstrated by Westerhausen et al (2016) and Schnitzler et al (2019). The decrease in size and polydispersity is most impressive in lipid nanoparticle formulations with siRNA Krzysztoń et al, 2017). They all show that increasing mixing speeds decrease size and polydispersity.…”

Section: Discussionmentioning

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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“…Continuous or batch wise production of PEI pDNA polyplexes with active mixing by surface acoustic waves (SAWs) has been demonstrated by Westerhausen et al (Westerhausen et al, 2016) and Schnitzler et al (Schnitzler et al, 2019). The decrease in size and polydispersity is most impressive in lipid nanoparticle formulations with siRNA (Chen et al, 2012;Krzysztoń et al, 2017). They all show that increasing mixing speeds decrease size and polydispersity.…”

Section: Discussionmentioning

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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Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles

Cited by 60 publications

References 44 publications

State‐of‐the‐Art Design and Rapid‐Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

State‐of‐the‐Art Design and Rapid‐Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

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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