Journal: Genes & Diseases

Article Title: A novel aptamer-based small RNA delivery platform and its application to cancer therapy

doi: 10.1016/j.gendis.2022.05.004

Figure Lengend Snippet: Magnesium induced assembly of nanoparticles with improved resistance to degradation and miRNA function. (A) Micelle formation under various concentrations of MgCl 2 detected by hydrophobic-incorporation of fluorescent dye (CMC-535). (B) Plots of fluorescence intensity at 535 nm under various concentrations of the delivery platform. The critical micelle concentration (CMC) was 3 × 10 -7 M. (C) Transmission electron microscopy image of the delivery platform nanoparticles using negative staining. Scale bar, 50 nm. Representative image of a single experiment. (D) The particle size distribution of the delivery platform with MgCl 2 (+Mg 2+ ) or without MgCl 2 (-Mg 2+ ) measured by dynamic light scattering. Representative image of two independent experiments. (E) Stability of each component of the miR-26a delivery platform against serum degradation in vitro . The miR-26a chimera of particle form (+Mg 2+ ) or non-particle monomer form (-Mg 2+ ) in various formats ( Fig. S3A ) was incubated with human serum for various time periods. Stability was measured by qPCR for miR-26a. (F) Functional effect of each moiety in the delivery platform for gene silencing by miR-26a. The c-Kit-targeting miR-26a chimera (1 μM) in various formats ( Fig. S3A ) with or without MgCl 2 were incubated with c-Kit + TUBO cancer cells for 2 days. The expression levels of a miR-26a target gene, Ezh2 , were measured by qPCR. Asterisks denote the significant difference compared to vehicle controls. (A, B, E, F) Data shown as mean ± SD of combined data from two independent experiments, each with duplicated samples. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Fluorescence spectroscopy was used to estimate the critical micelle concentration (CMC) of the delivery platform using a hydrophobic fluorescent probe following the manufacturer's protocol (CMC-535 detergent assay, G-Bioscience, St. Louis, MO) at 25 °C.

Techniques: Fluorescence, Concentration Assay, Transmission Assay, Electron Microscopy, Negative Staining, In Vitro, Incubation, Functional Assay, Expressing

Journal: Genes & Diseases

Article Title: A novel aptamer-based small RNA delivery platform and its application to cancer therapy

doi: 10.1016/j.gendis.2022.05.004

Figure Lengend Snippet: The pH-sensitive delivery platform induced endosomal leakage but not cytotoxicity. (A) Target specificity of the c-Kit-targeting delivery platform linked with anti-c-Kit DNA aptamer. The c-Kit-targeting aptamer-positive platform bound specifically to c-Kit + MEF cells in vitro . (B) Visualizing uptake of c-Kit-targeting miR-26a chimera in c-Kit + MEF cells over 120 min. The AF488-conjugated miR-26a chimera (Green) was incubated with the cells at 37 °C. After washing with PBS, the cells were fixed with 4% formaldehyde and images were acquired on a fluorescent microscope. Scale bar, 5 μM. (C) Destabilization of micelle particles of the miR-26a chimera under acidic pH conditions detected by hydrophobic-incorporation of fluorescent dye (CMC-535). Tween-20 is a used as a control. Asterisks denote significant differences compared to pH 7.0. (D) Free magnesium concentration in solution of the delivery platform nanoparticles incubated under various pH conditions for 1 h. Asterisks denote significant differences compared to pH 7.0. (E) Release of an endosomal trafficking fluorescent probe (10 k Dextran, red) from endosomes in c-Kit + MEF cells at 3 h after treatment with vehicle or miR-26a chimera (Top panels). Release of another fluorescent probe (Cathepsin enzymatic fluorescent substrate, Magic Red) from endosomes in c-Kit + MEF cells at 3 h after treatment with vehicle or miR-26a chimera (Bottom panels). Scale bar, 5 μM. (F) Lack of cytotoxicity of miR-26a chimera to cKit + MEF cells treated with various concentration of miR-26a chimera for 24 h as detected by LDH release assay. Asterisks denote the significant difference compared to 0 μM of miR-26a chimera treatment. (A, B, E) Representative images of two independent experiments. (C, D, F) Data shown as mean ± SD of combined data from two independent experiments, each with duplicated samples. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Fluorescence spectroscopy was used to estimate the critical micelle concentration (CMC) of the delivery platform using a hydrophobic fluorescent probe following the manufacturer's protocol (CMC-535 detergent assay, G-Bioscience, St. Louis, MO) at 25 °C.

Techniques: In Vitro, Incubation, Microscopy, Control, Concentration Assay, Lactate Dehydrogenase Assay