Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.
Last. Pieter R. Cullis(UBC: University of British Columbia)H-Index: 117
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The regulatory approval of Onpattro, a lipid nanoparticle-based short interfering RNA drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, paves the way for clinical development of many nucleic acid-based therapies enabled by nanoparticle delivery.
Last. Pieter R. Cullis(UBC: University of British Columbia)H-Index: 117
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Onpattro, the first RNAi-based therapeutic to receive FDA approval, is enabled by a lipid nanoparticle (LNP) system that facilitates siRNA delivery into the cytoplasm of target cells (hepatocytes) following intravenous (i.v.) administration. These LNP-siRNA systems consist of four lipid components (ionizable cationic lipid, distearolyphosphatidycholine or DSPC, cholesterol, and PEG-lipid) and siRNA. The ionizable cationic lipid has been optimised for RNA encapsulation and intracellular delivery,...
Last. Roy van der Meel(TU/e: Eindhoven University of Technology)H-Index: 21
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ConspectusDelivering nucleic acid-based therapeutics to cells is an attractive approach to target the genetic cause of various diseases. In contrast to conventional small molecule drugs that target gene products (i.e., proteins), genetic drugs induce therapeutic effects by modulating gene expression. Gene silencing, the process whereby protein production is prevented by neutralizing its mRNA template, is a potent strategy to induce therapeutic effects in a highly precise manner. Importantly, gen...
Last. Pieter R. Cullis(UBC: University of British Columbia)H-Index: 117
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The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. ...
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 endonucleas...
#2Pieter R. Cullis(UBC: University of British Columbia)H-Index: 117
Last. Roy van der Meel(UBC: University of British Columbia)H-Index: 21
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Genetic drugs based on RNA or DNA have remarkable therapeutic potential as virtually any disease can be treated by silencing a pathological gene, expressing a beneficial protein, or by editing defective genes. However, therapies based on nucleic acid polymers require sophisticated delivery systems to deliver these macromolecules to the interior of target cells. In this study, we review progress in developing nonviral lipid nanoparticle (LNP) delivery systems that have attractive properties, incl...
#2Maria M. Darjuan(UBC: University of British Columbia)H-Index: 2
Last. Pieter R. Cullis(UBC: University of British Columbia)H-Index: 117
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Lipid nanoparticles (LNPs) containing short interfering RNA (LNP-siRNA) and optimized ionizable cationic lipids are now clinically validated systems for silencing disease-causing genes in hepatocytes following intravenous administration. However, the mechanism of formation and certain structural features of LNP-siRNA remain obscure. These systems are formed from lipid mixtures (cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid) dissolved in ethanol that is rapidly mixed w...
: Lipid nanoparticles (LNPs) are established in the biopharmaceutical industry for efficient encapsulation and cytosolic delivery of nucleic acids for potential therapeutics, with several formulations in clinical trials. The advantages of LNPs can also be applied in basic research and discovery with a microfluidic method of preparation now commercially available that allows preparations to be scaled down to quantities appropriate for cell culture. These preparations conserve expensive nucleic ac...
#2Yuen Yi C. Tam(UBC: University of British Columbia)H-Index: 23
Last. Souvik Biswas(UBC: University of British Columbia)H-Index: 13
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A straightforward “bottom-up” synthesis is described for efficient entrapment of inorganic hydrophobic nanoparticles (HNPs) consisting of iron oxide, gold, or quantum dots within the hydrophobic core of lipid nanoparticles (LNPs). These LNPs consist of hydrophobic “core” lipids such as triolein surrounded by a monolayer of amphipathic “surface” lipids, such as phosphatidylcholine and polyethylene-glycol-lipid. It is shown that rapid, controlled mixing of HNPs, core lipids and surface lipids in a...
Last. Pieter R. Cullis(UBC: University of British Columbia)H-Index: 117
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Abstract Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo . Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro ...
#1Amrita Das(IICB: Indian Institute of Chemical Biology)H-Index: 15
#2Nahid Ali(IICB: Indian Institute of Chemical Biology)H-Index: 33
Vaccination is so far the most effective way of eradicating infections. Rapidly emerging drug resistance against infectious diseases and chemotherapy-related toxicities in cancer warrant immediate ...
Lipid nanoparticles (LNPs) have emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutics. Currently in the spotlight as vital components of the COVID-19 mRNA vaccines, LNPs play a key role in effectively protecting and transporting mRNA to cells. Liposomes, an early version of LNPs, are a versatile nanomedicine delivery platform. A number of liposomal drugs have been approved and applied to medical practice. Subsequent generations of lipid nanocarrier...
Abstract null null In 2018 I was appointed full professor of Pharmaceutical Biotechnology & Delivery at the Pharmaceutics division of the department of Pharmaceutical Sciences at Utrecht University, The Netherlands. In this contribution to the Orations – New Horizons of the Journal of controlled Release I will introduce my research group (see also null www.uu.nl/pharmaceutics ) and will highlight my current and future research projects. In coming years the focus of my research will be on the inj...
Vaccination represents the best line of defense against infectious diseases and is crucial in curtailing pandemic spread of emerging pathogens to which a population has limited immunity. In recent years, mRNA vaccines have been proposed as the new frontier in vaccination, owing to their facile and rapid development while providing a safer alternative to traditional vaccine technologies such as live or attenuated viruses. Recent breakthroughs in mRNA vaccination has been through formulation with ...
Genome-wide association studies have shown that a gene variant in the Family with Sequence Similarity 13, Member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with Chronic Obstructive Pulmonary Disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor β1 (TGFβ1). To determine the role of FAM13A in TGFβ1 signaling, FAM13A-/- airway epithelial cells were generated using...
Last. Yuen Yi C. Tam(UBC: University of British Columbia)H-Index: 23
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Lipid-based formulations have been developed to improve stability profiles, tolerability, and toxicity profiles of small molecule drugs. However, manufacture of such formulations involving lipophilic compounds can be labor-intensive and difficult to scale because of solubility and solvent compatibility issues. We have developed a rapid and scalable approach using rapid-mixing techniques to generate homogeneous lipid nanoparticle (LNP) formulations of siRNA, triglycerides, and hydrophilic weak-ba...
Chitosan-based carriers have coined their position as delivery agents. When assembled with polyanions into nanogels (NG), these vectors have enabled the delivery of drugs, genes, and proteins to a myriad of applications. However, the chemical and colloidal instability of chitosan nanoformulations in physiologically compatible media prejudices in vitro biocompatibility and, thus, scale-up applications. To overcome this issue, we envisaged the coating of chitosan nanogel with phospholipids. In thi...
The prior year has seen an unprecedented number of vaccine candidates directed at the COVID-19 pandemic. This report examines these vaccines and the related research effort, both traditional and fo...
