Volume 25, Issue 7, Pages 1544-1555 (July 2017) RNA Nanoparticle-Based Targeted Therapy for Glioblastoma through Inhibition of Oncogenic miR-21  Tae Jin Lee, Ji Young Yoo, Dan Shu, Hui Li, Jianying Zhang, Jun-Ge Yu, Alena Cristina Jaime-Ramirez, Mario Acunzo, Giulia Romano, Ri Cui, Hui-Lung Sun, Zhenghua Luo, Matthew Old, Balveen Kaur, Peixuan Guo, Carlo M. Croce  Molecular Therapy  Volume 25, Issue 7, Pages 1544-1555 (July 2017) DOI: 10.1016/j.ymthe.2016.11.016 Copyright © 2016 Terms and Conditions

Figure 1 Construction and Characterization of the Multifunctional FA-3WJ-Alexa647-LNA-miR21 RNP for Targeted Therapy of Glioblastoma (A) Sequence map of the trivalent FA-3WJ-Alexa647-LNA-miR21 RNP constructed from four module strands, of which three individual strands were to harbor: FA as a targeting ligand; Alexa647 as an imaging module; and anti-miR-21 LNA for miR-21 knockdown. (B) AFM image showing three-branched triangular structure of self-assembled trivalent FA-3WJ-Alexa647-LNA-miR21 RNP. To make the FA-3WJ-Alexa647-LNA-miR21 RNP to be within suitable persistence length (stiffness) for AFM imaging, overall length of RNP was extended by attaching 58–60 bp dsRNA to each arm strand of the RNP. Although the extension exaggerated the observed size of RNPs in the AFM image more than their original sizes, the shape of the AFM image was expected to reflect the three-branched global structure of 3WJ core-based FA-3WJ-Alexa647-LNA-miR21 RNPs. (C) The serum stability of FA-3WJ-Alexa647-LNA-miR21 RNP was incubated in 50% of FBS at 37°C for up to 24 hr. The arrowhead indicates the intact form of the self-assembled FA-3WJ-Alexa647-LNA-miR21 RNP. (D) The DLS data illustrating the size of FA-3WJ-Alexa647-LNA-miR21 RNP is shown. (E) The zeta potential of FA-3WJ-Alexa647-LNA-miR21 RNP is shown. The data in (D) and (E) were obtained from three independent experiments. Molecular Therapy 2017 25, 1544-1555DOI: (10.1016/j.ymthe.2016.11.016) Copyright © 2016 Terms and Conditions

Figure 2 FA-Dependent Specific Targeting of Human Glioblastoma Cells by FA-3WJ-Alexa647-LNA-miR21 RNP In Vitro (A) Flow cytometric analysis on human glioblastoma cell U87EGFRvIII after incubation with FA-3WJ-Alexa647-LNA-miR21 RNP. The fluorescence intensity from the Alexa647 fluorophore of FA-3WJ-Alexa647-LNA-miR21 RNPs in association with U87EGFRvIII cells was compared to a negative control RNP (FA-free 3WJ-Alexa647 RNP) and PBS. The percentage of Alexa647 positive cells was averaged from three independent experiments and analyzed by Student’s t test (p < 0.001 and n = 3). (B) The representative fluorescence confocal microscope image at 60× magnification illustrating the FA-dependent and specific targeting, as well as the intracellular distribution of the FA-3WJ-Alexa647-LNA-miR21 RNP in U87EGFRvIII cells is shown. The pseudocolor was used for nucleus identification (blue), cytoskeleton (green), and Alexa647 (red). (C) The fluorescence live cell confocal microscopy illustrating the real-time events during the specific FA-dependent association between FA-3WJ-Alexa647-LNA-miR21 RNPs and U87EGFRvIII cells in comparison to the negative control RNP, 3WJ-Alexa647 is shown. The live confocal images were taken at 20× magnification every 10 s beginning at time 0 after the addition of RNPs up to 32 min. Pseudocolor for Alexa647 (red) was used. The scale bars represent 20 μm. Whole time images were presented in Movies S1 and S2. Molecular Therapy 2017 25, 1544-1555DOI: (10.1016/j.ymthe.2016.11.016) Copyright © 2016 Terms and Conditions

Figure 3 Specific FA-Dependent Knock Down of miR-21 by FA-3WJ-Alexa647-LNA-miR21 RNPs in Human Glioblastoma Cells (A) Endogenous expression levels of miR-21 were assessed in U87EGFRvIII cells treated with FA-3WJ-Alexa647-LNA-miR21 RNPs via TaqMan microRNA expression assays using real-time qPCR. (B) Dual luciferase assays determined the specificity of FA-3WJ-Alexa647-LNA-miR21 RNPs against mature miR-21 sequences as compared to FA-free negative control RNPs. (C) The cytotoxicity in U87EGFRvIII cells treated with a wide range (0 to 1,000 nM) of FA-3WJ-Alexa647-LNA-miR21 RNPs (red circles) or FA-3WJ-Alexa647-LNA-SC RNPs (negative control, blue circles) (n = 6) is shown. The cell viabilities were evaluated by MTS assay. The FA-dependent specificity was tested in the presence or absence of 1 mM free folate in the culture media. (D and E) The flow cytometry for apoptotic cell detection by annexin V and PI double staining (D) and by cell cycle analysis via PI staining (E) was performed on U87EGFRvIII cells treated with FA-3WJ-Alexa647-LNA-miR21 RNPs or FA-3WJ-Alexa647-LNA-SC RNPs (negative control). Annexin V positive cells were considered to be apoptotic cells. The PBS-treated U87EGFRvIII cells were used as gating controls. All of the data were obtained from three individual experiments and analyzed by a Student’s t test. All of the error bars indicate SEM. *p < 0.01; **p < 0.05; ***p > 0.05. Molecular Therapy 2017 25, 1544-1555DOI: (10.1016/j.ymthe.2016.11.016) Copyright © 2016 Terms and Conditions

Figure 4 Specific FA-Dependent FA-3WJ-Alexa647-LNA-miR21 RNPs Targeting of Human Glioblastoma Patient-Derived Cell GBM30 In Vivo (A) Representative ex vivo MRI images illustrate tumor locations and size (left). Following systemic administration of FA-3WJ-Alexa647-LNA-miR21 RNPs, FA-dependent tumor targeting was visualized via fluorescence ex vivo imaging in comparison to FA-free or FA-conjugated 3WJ-Alexa647 RNPs (right). The PBS-injected mouse brains were used to determine background auto-fluorescence intensity. (B) The mean fluorescence intensity was calculated from the average fluorescence intensity divided by tumor volume (mm3) and used to normalize the variation among the tested mice. All of the error bars indicate SEM, and a Student’s t test was used for statistical analysis. *p < 0.01 (n = 3). (C) The biodistribution profile of FA-3WJ-Alexa647-LNA-miR21 RNPs was obtained by examining fluorescence signals on major internal organs collected from the RNP-injected mice. Molecular Therapy 2017 25, 1544-1555DOI: (10.1016/j.ymthe.2016.11.016) Copyright © 2016 Terms and Conditions

Figure 5 Targeted Therapy of Human Glioblastoma Cells following the Systemic Delivery of FA-3WJ-Alexa647-LNA-miR21 RNPs (A) Animal study scheme shows overall timeline of the in vivo experiment for the RNP-based targeted therapy of human glioblastoma in mice. A total of five dosages of RNPs were injected on every other day starting at day 7 from the tumor implantation (day 0). The bioluminescence whole body imaging was taken on 1 day after each RNP injection. (B) The tumor regression was observed following the repeated systemic delivery of FA-3WJ-Alexa647-LNA-miR21 RNPs, which was determined via luciferase monitoring of luciferase-expressing GBM30-Luc cells in vivo following five RNP injections. All of the error bars indicate SEM, and a Student’s t test was used for statistical analysis. *p < 0.01 (n = 5). (C) The representative bioluminescence image after the final injection of FA-3WJ-Alexa647-LNA-miR21 RNP compared to FA-3WJ-Alexa647-LNA-SC is shown. (D) Kaplan-Meier survival curves are shown in the image. *p < 0.01 (n = 5). (E) The TaqMan microRNA expression analysis via qreal-time PCR for endogenous miR-21 expression levels in tumor regions of mice treated with FA-3WJ-Alexa647-LNA-miR21 RNPs as compared FA-3WJ-Alexa647-LNA-SC RNPs is shown. **p < 0.05 (n = 3). (F) Western blotting on cell lysates from tumor regions of mice treated with FA-3WJ-Alexa647-LNA-miR21 RNPs as compared to FA-3WJ-Alexa647-LNA-SC RNPs for miR-21 targets and effector genes involved in cellular proliferation and apoptosis pathways is shown. (G) The immunohistochemistry on paraffin-embedded tumor tissue sections of mice treated with RNPs are shown. H&E and anti-Ki67 staining illustrates the tumor region with actively proliferating cells. A TUNEL assay was used to visualize the apoptotic cell population. Tumor, glioblastoma-induced xenograft tumor region; and PN, paired normal region. Molecular Therapy 2017 25, 1544-1555DOI: (10.1016/j.ymthe.2016.11.016) Copyright © 2016 Terms and Conditions