The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid‐Based Nanoparticles for mRNA and DNA Delivery

Kafetzis, Konstantinos N; Papalamprou, Natalia; McNulty, Elisha; Thong, Kai Xin; Sato, Yusuke; Mironov, Aleksandr; Purohit, Atul; Welsby, Philip J.; Harashima, Hideyoshi; Yu‐Wai‐Man, Cynthia; Tagalakis, Aristides D.

doi:10.1002/adhm.202203022

Cited by 16 publications

(11 citation statements)

References 85 publications

(152 reference statements)

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“…Additionally, the pH and ionic strength of the formulation buffer can affect vector transfection efficiencies. Gene therapy formulations are often stored by freezing or refrigeration which can increase temperature before delivery and reduce efficacy ( Kafetzis et al, 2023 ).…”

Section: Safety and Efficacy Issues Of Design Moleculesmentioning

confidence: 99%

Designing molecules: directing stem cell differentiation

Thanaskody,

Natashah,

Nordin

et al. 2024

Front. Bioeng. Biotechnol.

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Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

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Section: Safety and Efficacy Issues Of Design Moleculesmentioning

confidence: 99%

Designing molecules: directing stem cell differentiation

Thanaskody,

Natashah,

Nordin

et al. 2024

Front. Bioeng. Biotechnol.

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“…There are a limited number of published studies on freezing iLNPs. Sugar‐based cryoprotectants, such as sucrose, mannitol and trehalose have been reported, and the selection of buffers and freezing conditions have been investigated (Ball et al., 2017; Henderson et al, 2022; Kafetzis et al., 2023; Kim et al., 2023; Zhao et al., 2020). The optimal conditions reported for freezing mRNA‐iLNPs appears to be variable, depending on the ionizable lipid or lipidoid and RNA cargo.…”

Section: Commentarymentioning

confidence: 99%

mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles

McKenzie,

Minnell,

Ganley

et al. 2023

Current Protocols

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AbstractmRNA vaccines have recently generated significant interest due to their success during the COVID‐19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA‐iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA‐iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin‐MC3‐DMA. DLin‐MC3‐DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA‐iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailingBasic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticlesAlternate Protocol: Small‐scale encapsulation of mRNA using preformed vesiclesBasic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles

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“…NPs can also be coated with different modalities to increase their biocompatibility and enable higher NP concentrations to permeate the BBB. One NP coating option that has shown positive results is poly(ethylene glycol) (PEG) (Kafetzis et al, 2023). PEG has a dense near neutral composition enabling particles to 'experience' the brain ECs as a fluid rather than an impermeable solid.…”

Section: Caveolin-mediated Endocytosismentioning

confidence: 99%

Blood–brain barrier disruption in dementia: Nano‐solutions as new treatment options

Cooper,

Kafetzis,

Patabendige

et al. 2023

Eur J of Neuroscience

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Candidate drugs targeting the central nervous system (CNS) demonstrate extremely low clinical success rates, with more than 98% of potential treatments being discontinued due to poor blood–brain barrier (BBB) permeability. Neurological conditions were shown to be the second leading cause of death globally in 2016, with the number of people currently affected by neurological disorders increasing rapidly. This increasing trend, along with an inability to develop BBB permeating drugs, is presenting a major hurdle in the treatment of CNS‐related disorders, like dementia. To overcome this, it is necessary to understand the structure and function of the BBB, including the transport of molecules across its interface in both healthy and pathological conditions. The use of CNS drug carriers is rapidly gaining popularity in CNS research due to their ability to target BBB transport systems. Further research and development of drug delivery vehicles could provide essential information that can be used to develop novel treatments for neurological conditions. This review discusses the BBB and its transport systems and evaluates the potential of using nanoparticle‐based delivery systems as drug carriers for CNS disease with a focus on dementia.

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The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid‐Based Nanoparticles for mRNA and DNA Delivery

Cited by 16 publications

References 85 publications

Designing molecules: directing stem cell differentiation

Designing molecules: directing stem cell differentiation

mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles

Blood–brain barrier disruption in dementia: Nano‐solutions as new treatment options

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