Efficient delivery of siRNA to cortical neurons using layered double hydroxide nanoparticles (original) (raw)
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Journal of pharmaceutical sciences, 2013
The application of gene and RNAi-based therapies to the central nervous system (CNS), for neurological and neurodegenerative disease, offers immense potential. The issue of delivery to the target site remains the single greatest barrier to achieving this. There are challenges to gene and siRNA (small interfering RNA) delivery which are specific to the CNS, including the post-mitotic nature of neurons, their resistance to transfection and the blood–brain barrier. Viral vectors are highly efficient and have been used extensively in pre-clinical studies for CNS diseases. However, non-viral delivery offers an exciting alternative. In this review, we will discuss the extracellular and intracellular barriers to gene and siRNA delivery in the CNS. Our focus will be directed towards various non-viral strategies used to overcome these barriers. In this regard, we describe selected non-viral vectors and categorise them according to the barriers that they overcome by their formulation and targeting strategies. Some of the difficulties associated with non-viral vectors such as toxicity, large-scale manufacture and route of administration are discussed. We provide examples of optimised formulation approaches and discuss regulatory hurdles to clinical validation. Finally, we outline the components of an “ideal” formulation, based on a critical analysis of the approaches highlighted throughout the review. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
Biomaterials, 2014
Progression of RNA interference (RNAi)-based gene silencing technologies for the treatment of disorders of the central nervous system (CNS) depends on the availability of efficient non-toxic nanocarriers. Despite advances in the field of nanotechnology undesired and non-specific interactions with different brain-cell types occur and are poorly investigated. To this end, we studied the cytotoxic and neuroinflammatory effects of widely-used transfection reagents and modified amphiphilic βcyclodextrins (CDs). All non-viral vectors formed positively charged nanoparticles with distinctive physicochemical properties. Differential and significant cytotoxic effects were observed among commercially available cationic vectors, whereas CDs induced limited disruptions of cellular membrane integrity and mitochondrial dehydrogenase activity. Interestingly, murine derived BV2 microglia cells and a rat striatal in vitro model of Huntington's Disease (ST14A-HTT120Q) were more susceptible to toxicity than human U87 astroglioma cells. BV2 microglia presented significant increases in cytokine, toll-like receptor 2 and cyclooxygenase-2 gene expression after transfection with selected commercial vectors but not with CD.siRNA nanoparticles. Non-viral short interfering RNA (siRNA) nanoparticles formulated with G6 polyamidoamine (PAMAM) dendrimers also significantly increased cytokine gene expression in the brain following injections into the mouse striatum. Together our data identify modified CDs as nanosystems that enable siRNA delivery to the brain with low levels of cytotoxicity and immunological activation.
Nanomedicine: Nanotechnology, Biology and Medicine, 2019
Therapies to lower gene expression in brain disease currently require chronic administration into the cerebrospinal fluid (CSF) by intrathecal infusions or direct intracerebral injections. Though well-tolerated in the short-term, this approach is not tenable for a lifetime of administration. Nose-to-brain delivery of enriched chitosan-based nanoparticles loaded with anti-HTT siRNA was studied in a transgenic YAC128 mouse model of Huntington's Disease (HD). A series of chitosanbased nanoparticle (NP) formulations encapsulating anti-HTT small interfering RNA (siRNA) were designed to protect the payload from degradation "en route" to the target. Factors to improve production of effective nanocarriers of anti-HTT siRNA were identified and tested in a YAC128 mouse model of Huntington's disease. Four formulations of nanocarriers were identified to be effective in lowering HTT mRNA expression by at least 50%. Intranasal administration of nanoparticles carrying siRNA is a promising therapeutic alternative for safe and effective lowering of mutant HTT expression.
ACS omega, 2018
Layered double hydroxide (LDH) nanoparticles (NPs) are safe and effective vectors for small interfering RNA (siRNA) delivery. However, it is unclear whether there are optimal parameters for the efficient delivery of functional siRNA using LDH NPs. In this research, we comprehensively examined the effect of parameters, such as the mixing method and LDH/siRNA mass ratio on siRNA silencing capability. We first noted that the best way for loading gene segments (25 bp dsDNA and siRNA) is to add gene molecules to 100 nm LDH and then diluting in Dulbecco's modified Eagle's medium. Very interestingly, the optimal LDH/gene mass ratio is around 20:1 in terms of cellular uptake amount of gene segments, whereas this ratio is shifted to around 5:1 in terms of target gene silencing efficacy, which has been reasonably explained. The optimization for LDH NP-based gene delivery system may provide the guidance for more efficient in vitro and in vivo siRNA delivery using the optimal parameters.
assisted with the optimisation of genotyping protocols. Chapter III Dr. Susan Grenham assisted with the design of qPCR probes. Ms. Sheila O'Loughlin and Mr. Dinesh Rasiah assisted with the in vitro work in HD fibroblasts and qPCR analyses. Chapter IV Dr. Caroll J Beltran assisted with western blots. Dr. Cristina Torres-Fuentes assisted with HCA experiments. Mr. David J McCarthy assisted with in vitro work in U87 cells and qPCR analyses. Ms. Aoife Quinlan assisted with stereotaxic brain surgeries. Chapter V Ms. Aoife Quinlan assisted with pharmacokinetic study. Signed, ________________________ Bruno MDC Godinho x Acknowledgments Firstly, I wish to thank my supervisors Prof. John Cryan and Prof. Caitriona O'Driscoll for the oportunity of conducting my research under their guidance and supervision at School of Pharmacy and the department of Anatomy and Neuroscience. Their vast expertise and knowledge of the field were key for the achievements of this project. In addition, personally both were source of great inspiration and motivation, further developing my passion for science and research. Carrying out my PhD studies would not have been possible without the support of many others that somehow have helped and enabled me to achieve the set goals.
Nano Today, 2015
RNAi has always captivated scientists due to its tremendous power to modulate the phenotype of living organisms. This natural and powerful biological mechanism can now be harnessed to downregulate specific gene expression in diseased cells; opening up endless opportunities. Since most of the conventional siRNA delivery methods are limited by a narrow therapeutic index and significant side and off-target effects, we are now in the dawn of a new age in gene therapy driven by nanotechnology vehicles for RNAi therapeutics. Here, we outlook the "do's and dont's" of the *Manuscript Click here to view linked References REVIEW NANO TODAY inorganic RNAi nanomaterials developed in the last 15 years and the different strategies employed are compared and scrutinized, offering important suggestions for the next 15.
Peptide-siRNA Supramolecular Particles for Neural Cell Transfection
Advanced Science, 2018
The development of efficient, easy-touse and nontoxic molecular carriers for delivery of small interfering ribonucleic acid (siRNA) into neural cells (neurons and glial cells) would accelerate the functional screening of genes and consequent discovery of therapeutic targets for neurodegenerative diseases. [1-4] Transfection of primary neuronal cells, however, is challenging in part due to low survival rate in vitro and their lower nanoparticle uptake compared to neuronal cell lines. [5] At the same time, there is a lack of appropriate nonviral methods that integrate high transfection efficiency, low cytotoxicity, biodegradability, and scalability. [6,7] Peptides have been of interest as siRNA delivery systems since they are structurally well defined, biodegradable, often nontoxic, and can be prepared through cost-effective Small interfering ribonucleic acid (siRNA)-based gene knockdown is an effective tool for gene screening and therapeutics. However, the use of nonviral methods has remained an enormous challenge in neural cells. A strategy is reported to design artificial noncationic modular peptides with amplified affinity for siRNA via supramolecular assembly that shows efficient protein knockdown in neural cells. By solid phase synthesis, a sequence that binds specifically double-stranded ribonucleic acid (dsRNA) with a selfassembling peptide for particle formation is integrated. These supramolecular particles can be further functionalized with bioactive sequences without affecting their biophysical properties. The peptide carrier is found to silence efficiently up to 83% of protein expression in primary astroglial and neuronal cell cultures without cytotoxicity. In the case of neurons, a reduction in electrical activity is observed once the presynaptic protein synaptophysin is downregulated by the siRNA-peptide particles. The results demonstrate that the supramolecular particles offer an siRNA delivery platform for efficient nonviral gene screening and discovery of novel therapies for neural cells.
Characterization and comparison of two novel nanosystems associated with siRNA for cellular therapy
International journal of pharmaceutics, 2016
To direct stem cell fate, a delicate control of gene expression through small interference RNA (siRNA) is emerging as a new and safe promising strategy. In this way, the expression of proteins hindering neuronal commitment may be transiently inhibited thus driving differentiation. Mesenchymal stem cells (MSC), which secrete tissue repair factors, possess immunomodulatory properties and may differentiate towards the neuronal lineage, are a promising cell source for cell therapy studies in the central nervous system. To better drive their neuronal commitment the repressor Element-1 silencing transcription (REST) factor, may be inhibited by siRNA technology. The design of novel nanoparticles (NP) capable of safely delivering nucleic acids is crucial in order to successfully develop this strategy. In this study we developed and characterized two different siRNA NP. On one hand, sorbitan monooleate (Span(®)80) based NP incorporating the cationic components poly-l-arginine or cationized p...