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Tools & Techniques: Quantum Dot-based microRNA Detection; siRNA Delivery to Leukemia Cells; and More


Although several microRNA detection techniques exist, they tend to be expensive and time-consuming, and are largely inapplicable for in situ or intracellular studies.

In order to meet the need for improved miRNA detection approaches, a team from Nanjing University has developed a novel quantum dot-based method to detect the small, non-coding RNAs that they say has potential for clinical applications.

In addition to PCR, microarray analysis, and Northern blotting approaches, miRNA detection methods based on alternative biosensing techniques, such as nanoparticle-amplified surface plasmon resonance imaging, have been developed, the researchers wrote in PLoS One.

However, these methods are limited by their requirement for miRNA enrichment followed by labeling of miRNA isolated from the sample prior to detection.

Previously, the team developed cellular delivery systems that used polyethylenimine-grafted graphene nanoribbons and multifunctional SnO2 nanoparticles as gene vectors for target cell-specific imaging and detection of intracellular pre-miRNA by fluorescence resonance energy transfer.

In the investigators’ latest work, they created miRNA probes by assembling thiolated RNA to gold nanoparticles, and binding the 3’ end amine of the RNA to the carboxy group on the surface of quantum dots, according to the PLoS One paper. The probes were assembled on gene vectors by mixing them with aqueous poly(γ-glutamic acid) solution and then with a solution of the biocompatible polymer chitosan.

After being delivered into living cells, the complex reacted to the intracellular pH and released the quantum dot-bound RNA probes, which hybridized with pre-miRNA precursors. The formed product was cleaved by Dicer, separating quantum dots and from the nanoparticles and triggering fluorescence emissions detectable by confocal microscopic imaging, which indicated the amount of intracellular pre-miRNA precursors.

Given the growing body of data linking miRNAs to disease, the new detection method offers a “new avenue for monitoring the intracellular pre-miRNA precursors, showing promising application in biomedicine,” the researchers concluded.

RNAi holds promise for treating a variety of diseases, including ones that affect the pulmonary epithelium. Still, the lack of efficient lung-delivery vectors, particularly for diseased tissue, has hampered the development of RNAi drugs in this space.

The majority of research aiming to deliver RNAi molecules to the lung has focused on intranasal or inhalation routes of administration, a team led by investigators at the University of Queensland Diamantina Institute wrote in Molecular Therapy — Nucleic Acids.

“However, this may be complicated in a diseased state due to the increased fluid production and tissue remodeling,” they noted. “In addition, tissue inflammation and extensive deposition of extracellular matrix might hamper delivery to the lower respiratory tract potentially limiting the therapeutic use of inhalation delivery systems.”

The scientists therefore explored the possibility of intravenously delivering liposome-formulated siRNAs to the lung epithelium, in light of data in the literature showing that such an approach can successfully lead to the knockdown of endothelial-specific genes.

The team tested cationic stealth liposomes, generated using a previously reported hydration of a freeze-dried matrix method, loaded with siRNAs. They found that, after intravenous injection, around 45 percent of epithelial murine lung cells received siRNA delivery, resulting in “targeted gene and protein knockdown throughout the lung, including lung epithelium.”

This is the first time lung epithelial delivery using cationic liposomes has been demonstrated, and the researchers cautioned that the exact mechanism behind the transfection remains unknown.

“However, one can envision that using this method may provide a more efficient means of treating diseased or infected lungs when compared to other routes of delivery,” they wrote, adding that they are currently examining the delivery method in pathological conditions including viral infections.

Although there are a number of computational tools for miRNA prediction, oftentimes these are pre-miRNA predictors that do not provide information about putative miRNA location within the pre-miRNA, according to a research team at McGill University.

“Sequence and structural features that determine the location of the miRNA, and the extent to which these properties vary from species to species, are poorly understood,” they wrote in Nucleic Acids Research.

Aiming to address this issue, the team developed miRdup, a computational predictor for the identification of the most likely miRNA location within a specific pre-miRNA, or for the validation of a candidate miRNA.

The tool is based on a random forest classifier trained with experimentally validated miRNAs from five lineages — mammals, fishes, arthropods, nematodes, and plants — which is designed to increase species specificity and allow for the discovery of features that distinguish miRNAs between species.

The algorithm works on both single hairpin and multiloop pre-miRNAs, the team wrote, and it automatically downloads and trains on the latest miRbase release to remain up to date. It is available here.

Despite its therapeutic potential, particularly for the treatment of cancers resistant to conventional therapy, RNAi suffers from a lack of effective delivery vehicles for difficult-to-treat cells.

Among these are chronic myeloid leukemia cells, and in an effort to overcome this hurdle, a team from the University of Alberta has developed lipophilic polymers capable of delivering siRNAs into such cancer cells.

Viral vectors have proven effective in manipulating leukemia cells, the researchers wrote in the Journal of Controlled Release, but they represent a safety risk due to their ability to integrate into a host genome or cause lethal immune responses.

Cationic polymers, meanwhile, are safer and are particularly amenable to engineering. In particular, lipid-substituted polyethylenimines, or PEIs, have been developed and “tailored to deliver plasmid DNA, as well as siRNA, to a variety of cell types,” they noted.

In their study, the team focused on low-molecular weight PEIs, which show low cytotoxicity compared with their high-molecular weight counterparts.

“By employing the amine groups of PEI for substitutions, we found that the relatively nontoxic but ineffective 2 kDa PEI polymer could be transformed into an effective nucleic acid carrier as a result of lipid substitution on these groups,” they wrote.

In particular, palmitic acid-substituted 1.2 kDa PEI was found to be “highly efficient” in delivering siRNA and silencing of a reporter gene in K562 used as a model for leukemia. Notably, the silencing efficacy achieved with this polymer was higher than 25 kDa PEI and is similar to the commercial reagent Lipofectamine 2000, according to the Journal of Controlled Release paper.

When siRNAs against BCR-ABL — a therapeutically relevant target for chronic myeloid leukemia — were delivered to the K562 cells, the investigators observed a reduction in the corresponding mRNA levels, as well as an induction of early- and late-stage apoptosis.

Overall, the delivery method may provide an effective delivery strategy for RNAi-based chronic myeloid leukemia treatment, the team concluded.