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Tools & Techniques: Gold Nanoparticles for siRNA Delivery; Improved microRNA Extraction; and More


A multi-institute team of Spanish and German researchers has developed functionalized gold nanoparticles capable of delivering siRNAs specifically into cancer cells in vivo.

To date, a number of groups have used such nanoparticles as efficient, non-toxic carriers for siRNAs, yet the majority of these have only been tested in cell cultures against reporter genes, the investigators wrote in Biomaterials.

Recently, they demonstrated an RNAi effect using various gold nanoparticles in three biological systems of increasing complexity — in vitro cultured human cells, in vivo freshwater polyp, and in vivo healthy nude mice — and pinpointed certain design parameters that were required for functionalizing the nanoparticles for use in different organisms.

For instance, only a covalent bond between the siRNA and the nanoparticle ensured active RNAi release in the mice, an efficiency that was reinforced by the addition of RGD peptide to the surface of the nanoparticle, they wrote.

Aiming to test their delivery approach in a disease setting, the scientists focused on lung cancer given that RGD is recognized by cell-surface receptors, such as integrins, that are involved in cell adhesion and are expressed on proliferating endothelial cells such as those found in tumors.

They delivered the gold/RGD nanoparticles loaded with siRNAs against c-Myc — a regulator gene commonly mutated in many cancers — intratracheally into a lung cancer syngeneic orthotopic murine model, and found that the treatment resulted in significant target gene downregulation followed by tumor growth inhibition and prolonged animal survival.

“These results reiterate the capability of functionalized gold nanoparticles for targeted delivery of siRNA to cancer cells towards effective silencing of the specific target oncogene,” they concluded. “What is more, we demonstrate that the gold nanoconjugates trigger a complex inflammatory and immune response that might promote the therapeutic effect of the RNAi to reduce tumor size with low doses of siRNA.”

Looking to improve the detection of disease-associated circulating microRNAs, a team of Chinese researchers has developed a biosensor capable of identifying cancer-related miRNAs in blood using DNA concatamer amplification.

“Many methods have been developed for analyzing miRNA expression, including Northern blotting, microarray-based detection, and quantitative polymerase chain reaction,” the scientists wrote in Biosensors and Bioelectronics. But these approaches, they said, are limited by their efficacy, need for specialized skills, cost, or complexity.

To overcome this, the team developed a sensitive electrochemical miRNA biosensor based on two auxiliary probes, which can self-assemble to form one-dimensional DNA concatamers that serve as carriers for signal amplification, they wrote in their paper.

To test the approach on miR-21, they chose hexaammineruthenium(III) chloride as a signal reporter since it can bind to negatively charged DNA strands and screen-printed gold electrodes as substrate for surface immobilization of capture probes.

“The capture probe comprises a loop component, which includes a sequence complementary to the target,” they stated. “In the absence of target miRNA-21, the capture probe exits predominantly in the hairpin form. The DNA concatamers cannot bind to the capture probe through the complementary base pairing.”

In this state, only small electrochemical signals were observed. However, in the presence of miR-21, the stem-loop structure of the capture probe is unfolded, allowing the DNA concatamers to hybridize with the terminus of the capture probe.

“Consequently, numerous redox indicators can be gathered to the working electrode via DNA concatamers and eventually produce an amplified electrochemical signal,” they added.

With the sensor, miR-21 was able to be detected at concentrations ranging from 100 aM to 100 pM, with a detection limit of 100 aM, in human serum.

Based on the method’s sensitivity, selectivity, and stability, the research group sees potential for its use in miRNA diagnostics and prognostics.

In order to improve the extraction of miRNAs from cells, a process that often results in RNA degradation, a group from the Korea Institute of Science has developed a new method that boosts the amount of the non-coding RNAs that can be obtained.

“Typically, the procedure of miRNA extraction involves three basic steps: lysis, organic extraction, and precipitation or separation,” they explained in a paper appearing in Analytical and Bioanalytical Chemistry. “Among these steps, lysis is the most basic and important step, and it is carried out with solutions that include detergents to break down the cellular membrane. Therefore, the use of a proper detergent is essential in order to maximize the extraction of miRNA from cells.”

The investigators performed miRNA extraction from HeLa and H9c2 cells using a combination of Triton X-100, a nonionic detergent used for membrane permeabilization and cell lysis, and Life Technology’s TRIzol RNA isolation reagent.

Focusing on miR-21, they found that the combination of the two chemicals resulted in approximately 1.9-fold greater extraction recovery than with TRIzol alone.

“This method can be adapted for the quantification of miRNAs in cells, tissues, and biofluids, without additional amplification,” they wrote. “In addition, the method can also be applied for the extraction of miRNAs from vesicles enclosed by membranes and/or multi-protein complexes like exosomes.”

In an effort to improve the use of RNAi in clinical settings, researchers from Chongqing University have developed a heat-based method for inducing gene silencing.

Although conditional RNAi would enable the activation of target gene silencing only when it is therapeutically necessary, current approaches do not allow for the regulation of the quantity and timing of siRNA expression, the scientists wrote in Biotechnology Progress.

To address this, they constructed a novel siRNA plasmid expression vector that is initiated by human HSP70, a heat shock protein that is upregulated by heat stress and certain toxic agents, and carries a heat-inducible siRNA expression cassette.

They tested the system using siRNAs targeting the gama-synuclein gene in breast cancer cells, and found that target silencing occurs at relatively low levels — around 10.1 percent — at 37 degrees Celsius. With heat induction to 43 degrees Celsius via microwaves or ultrasound, target inhibition increased to 69.4 percent. Notably, cancer cell proliferation was inhibited following heat shock.

“We believe that the heat-inducible siRNA expression system described in this study provides a promising gene therapy approach that might be usefully applied in the treatment of cancer and other diseases,” the researchers wrote.

With the need for optimized siRNAs to support the development of RNAi therapeutics, a team from the City of Hope has investigated the use of 5’ unlocked nucleic acid modifications to boost the gene-silencing oligos’ silencing capabilities.

The preferential selection of the antisense strand by cellular RNAi machinery has been shown to improve gene-silencing efficiency while mitigating off-target effects, the team wrote in Molecular Therapy — Nucleic Acids.

Previously, other investigators have reported a 2’,3’-seco-RNA chemical modification, dubbed unlocked nucleic acid, that thermodynamically destabilizes RNA duplexes but preserves the A-form helix of double-stranded RNA. Placement of this modification on the first or second position of an siRNA strand blocks its gene-silencing ability.

To extend this effect, the researchers tested the 5’ UNA modification on different siRNA sequences and used cell-based assays to demonstrate the abrogation of RISC loading as a consequence of the modification. They also applied the UNA modification to transform less-functional siRNAs and Dicer-substrate siRNAs targeting a small conserved region of the HIV transcript into improved oligos that can attenuate HIV.

The work, the team concluded, “highlights the utility of 5’ UNA siRNA modification in therapeutic contexts where siRNA sequence selection is constrained.”