Blood-brain barrier disruption in dementia: Nano-solutions as new treatment options.

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.

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

Lipid-based nanoparticles have recently shown great promise, establishing themselves as the gold standard in delivering novel RNA therapeutics. However, research on the effects of storage on their efficacy, safety, and stability is still lacking. Herein, the impact of storage temperature on two types of lipid-based nanocarriers, lipid nanoparticles (LNPs) and receptor-targeted nanoparticles (RTNs), loaded with either DNA or messenger RNA (mRNA), is explored and the effects of different cryoprotectants on the stability and efficacy of the formulations are investigated. The medium-term stability of the nanoparticles was evaluated by monitoring their physicochemical characteristics, entrapment and transfection efficiency, every two weeks over one month. It is demonstrated, that the use of cryoprotectants protects nanoparticles against loss of function and degradation in all storage conditions. Moreover, it is shown that the addition of sucrose enables all nanoparticles to remain stable and maintain their efficacy for up to a month when stored at -80 °C, regardless of cargo or type of nanoparticle. DNA-loaded nanoparticles also remain stable in a wider variety of storage conditions than mRNA-loaded ones. Importantly, these novel LNPs show increased GFP expression that can signify their future use in gene therapies, beyond the established role of LNPs in RNA therapeutics.

Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery.

The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.

Advances in exosome therapies in ophthalmology-From bench to clinical trial.

During the last decade, the fields of advanced and personalized therapeutics have been constantly evolving, utilizing novel techniques such as gene editing and RNA therapeutic approaches. However, the method of delivery and tissue specificity remain the main hurdles of these approaches. Exosomes are natural carriers of functional small RNAs and proteins, representing an area of increasing interest in the field of drug delivery. It has been demonstrated that the exosome cargo, especially miRNAs, is at least partially responsible for the therapeutic effects of exosomes. Exosomes deliver their luminal content to the recipient cells and can be used as vesicles for the therapeutic delivery of RNAs and proteins. Synthetic therapeutic drugs can also be encapsulated into exosomes as they have a hydrophilic core, which makes them suitable to carry water-soluble drugs. In addition, engineered exosomes can display a variety of surface molecules, such as peptides, to target specific cells in tissues. The exosome properties present an added advantage to the targeted delivery of therapeutics, leading to increased efficacy and minimizing the adverse side effects. Furthermore, exosomes are natural nanoparticles found in all cell types and as a result, they do not elicit an immune response when administered. Exosomes have also demonstrated decreased long-term accumulation in tissues and organs and thus carry a low risk of systemic toxicity. This review aims to discuss all the advances in exosome therapies in ophthalmology and to give insight into the challenges that would need to be overcome before exosome therapies can be translated into clinical practice.

Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts.

The master regulator of the fibrosis cascade is the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway, making it a key target for anti-fibrotic therapeutics. In the past, inhibitors and small interfering RNAs (siRNAs) targeting the MRTF-B gene have been deployed to counter fibrosis in the eye, with the latter showing promising results. However, the biggest challenge in implementing siRNA therapeutics is the method of delivery. In this study, we utilised the novel, pH-sensitive, cationic lipid CL4H6, which has previously demonstrated potent targeting of hepatocytes and endosomal escape, to safely and efficiently deliver an MRTF-B siRNA into human conjunctival fibroblasts. We prepared two lipid nanoparticle (LNP) formulations, incorporating targeting cleavable peptide cY in one of them, and measured their physicochemical properties and silencing effect in human conjunctival fibroblasts. Both proved to be non-cytotoxic at a concentration of 50 nM and effectively silenced the MRTF-B gene in vitro, with the targeting cleavable peptide not affecting the silencing efficiency [LNP with cY: 62.1% and 81.5% versus LNP without cY: 77.7% and 80.2%, at siRNA concentrations of 50 nM (p = 0.06) and 100 nM (p = 0.09), respectively]. On the other hand, the addition of the targeting cleavable peptide significantly increased the encapsulation efficiency of the LNPs from 92.5% to 99.3% (p = 0.0005). In a 3D fibroblast-populated collagen matrix model, both LNP formulations significantly decreased fibroblast contraction after a single transfection. We conclude that the novel PEGylated CL4H6-MRTF-B siRNA-loaded LNPs represent a promising therapeutic approach to prevent conjunctival fibrosis after glaucoma filtration surgery.

RNA therapeutics in ophthalmology - translation to clinical trials.

The use of RNA interference technology has proven to inhibit the expression of many target genes involved in the underlying pathogenesis of several diseases affecting various systems. First established in in vitro and later in animal studies, small interfering RNA (siRNA) and antisense oligonucleotide (ASO) therapeutics are now entering clinical trials with the potential of clinical translation to patients. Gene-silencing therapies have demonstrated promising responses in ocular disorders, predominantly due to the structure of the eye being a closed and compartmentalised organ. However, although the efficacy of such treatments has been observed in both preclinical studies and clinical trials, there are issues pertaining to the use of these drugs which require more extensive research with regards to the delivery and stability of siRNAs and ASOs. This would improve their use for long-term treatment regimens and alleviate the difficulties experienced by patients with ocular diseases. This review provides a detailed insight into the recent developments and clinical trials that have been conducted for several gene-silencing therapies, including ISTH0036, SYL040012, SYL1001, PF-04523655, Sirna-027, QR-110, QR-1123, QR-421a and IONIS-FB-LRX in glaucoma, dry eye disease, age-related macular degeneration, diabetic macular oedema and various inherited retinal diseases. Our aim is to explore the potential of these drugs whilst evaluating their associated advantages and disadvantages, and to discuss the future translation of RNA therapeutics in ophthalmology.

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

INTRODUCTION: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. METHODS: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. RESULTS: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and ßENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. CONCLUSION: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.