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Drug Delivery

Drug Delivery - Poster Session D

Poster X19 - Design of Anti-microRNA Nanotubes to Target Invasiveness of Glioblastoma Cells

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Glioblastoma (GBM) is a highly aggressive primary brain tumor characterized by rapid growth, invasiveness, heterogeneity, and a high propensity for relapse. Current treatments, including surgical resection, radiation therapy, and chemotherapy, offer limited effectiveness, with a low median survival rate and a high likelihood of relapse. This limited success is primarily due to tumor heterogeneity, the immunosuppressive tumor microenvironment (TME), and the blood-brain barrier (BBB), which hinders drug delivery to the tumor site.

Oncogenic microRNAs (oncomiRs) play a crucial role in cancer development and progression by regulating genes involved in cell proliferation, apoptosis, invasion, and metastasis. In GBM, overexpression of microRNA-21 (miR-21) is correlated with tumor grade and associated with late-stage glioma, contributing to proliferation, invasion, drug resistance, and anti-apoptosis of cancer cells. Elevated miR-21 in M2-polarized tumor-associated macrophages (TAMs) further supports the tumor’s immunosuppressive environment.

Antisense oligonucleotides (anti-miRs) are designed to degrade or block specific oncomiRs, restoring signaling pathways that control tumor growth and apoptosis. Our group is developing a novel strategy to deliver anti-miRs to GBM using nucleic acid nanotubes (NTs). We have demonstrated that NTs composed of amphiphiles with a ssDNA headgroup and a hydrophobic tail, cross the BBB in a GL261 mouse model, accumulate at the tumor site, and internalize in GBM cells and TAMs.

In this study, we have designed anti-miR-21 NTs that effectively reduce the invasiveness of GBM cells in 3D organoids, potentially leading to improved treatment outcomes.

Materials and

Methods:  Anti-miR NT Synthesis
Anti-miR-21 is conjugated to a hydrophobic tail following established protocol in our lab. The amphiphile product is purified using high-performance liquid chromatography (HPLC) and the molecular weight is verified via mass spectrometry.

NT Uptake Experiments in Cell Monolayers
Confocal microscopy is used to verify NT uptake, and flow cytometry to quantify NT association with U87 cells. Intracellular trafficking of NTs is measured with flow cytometry, using various endocytosis inhibitors. Interaction between NTs and GBM cells is visualized with TEM.

Extracellular Matrix (ECM) Hydrogel
A hydrogel containing high and low molecular weight hyaluronic acid (HMHA and LMHA) and chondroitin sulfate (CS) is synthesized and the hydrogel’s stiffness is measured using a rheometer.

Organoid Culture
GFP-expressing CT-2A GBM cells, CellTrace Violet labeled RAW 264.7 tumor-associated macrophages, and CellTrace Far Red labeled SIM-A9 microglia are seeded in the hydrogel ECM to form micron-sized GBM spheroids using the water-in-oil microdroplet technique. The organoids are imaged over subsequent days to monitor cells and characterize cell migration, invasiveness and the interaction between different cells.

Results, Conclusions, and Discussions: Anti-miR-21 NTs were synthesized, purified, and characterized in vitro. Micron-long NTs are shortened using probe-sonication, reaching a desired submicron length that will allow for accumulation at the GBM tumor and uptake by cells in the TME. The dimensions of NTs were visualized through cryogenic-transmission electron microscopy (cryo-TEM).

RT-qPCR confirmed that the anti-miR-21 sequences effectively inhibited miR-21 expression in U87 cells. In vitro assays on monolayer cell culture confirmed anti-miR-21 NT association and uptake by U87 cells.

In 3D culture, cells in GBM spheroids demonstrated cell invasion into the surrounding ECM. Treatment with anti-miR-21 NTs reduced cell invasion compared to controls, highlighting the anti-miR NTs' potential as a therapeutic strategy for GBM.

To further investigate the NT effects on GBM invasion, more advanced organoid models are needed. The aim is to co-culture GBM cells with TAMs and microglia in clinically relevant ratios, using optimized scaffolds that mimic the biomechanical properties of GBM tumors. We are currently exploring the use of a hydrogel ECM comprising of HMHA, LMHA and CS. The ratios of these components in the synthetic ECM correspond to the composition of GBM ECM as reported in the literature. The invasiveness of GBM cells from the ECM core is assessed using phase contrast imaging. By tracing the perimeter of GBM cells that have invaded the surrounding ECM, the invaded area is quantified. Fluorescence imaging and confocal microscopy is used to observe cell interactions in the organoids.

In conclusion, the development of anti-miR-21 NTs and advanced organoid models shows promising potential for targeted GBM therapy and drug screening. Literature indicates that numerous oncomiRs play a role in GBM progression, which can be targeted with anti-miR NTs. By further developing our optimized organoid design, we aim to create a tunable and reproducible GBM model for large-scale drug screening, enabling the assessment of various anti-miR NTs on GBM and other cancers.

Acknowledgements (Optional):