(G-248) Biomimetic Phototheranostic Nanoparticles for Specific Targeting and Treatment of Glioblastoma

Introduction:: Glioblastoma is one of the most invasive and aggressive forms of brain cancer, with a 5-year survival rate < 7%. Treatment and early diagnosis are difficult as most therapeutic/diagnostic agents cannot pass through the blood-brain barrier (BBB). Extracellular vesicles (EVs), in particular, exosomes, have a natural ability to pass through the BBB due to natural protein markers expressed on their surface. EVs have been investigated as gene and drug delivery vehicles. Still, due to their rapid clearance, low loading capacity, and difficult detection using non-invasive imaging techniques, they have not been FDA-approved. Prussian Blue nanoparticles (PBNPs) have great photo-conversion properties that are beneficial in theranostic uses but cannot pass through the BBB. Here, we present a novel method of coating PBNPs with glioblastoma cell (U-87) derived exosomes to allow for passive transport into the brain and specific targeting of the tumor. These hybrid particles (Exo:PB) can be detected using photoacoustic imaging in vivo and can be used for photothermal therapy within the brain.


Materials and Methods:: Initially, Exo:PB particles were made using a co-extrusion method with PBNPs and U-87 derived exosomes at room temperature. Following extrusion, particles are stained with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI) and washed 3 times with water at 12,000 rpm for 20 minutes. Final suspensions are stored in the fridge and have stability for up to 9 months. For cell-based experiments, 1 mg/mL of Exo:PB, PBNP, or U-87 derived exosomes were treated to plated U-87 cells and left to incubate for 24 hours. The following day cells were either washed and stained or treated with an 808 nm laser for 1 minute. For in vivo photothermal therapy experiments, 1 mg of particle was injected intratumorally in a subcutaneous brain tumor mouse and then exposed to an 808 nm laser for 5 minutes. In vivo targeting studies were done in an orthotopic brain tumor mouse model by injecting 2mg of either Exo:PB or PEG:PB into the tail vein. Photoacoustic images were taken pre-injection, immediately after injection, and at 1-, 3-, and 24-hour time points. All in vitro and in vivo studies were done with at least an N=3. Statistical analysis was done using a student’s t-test, and values were considered significant if p< 0.05.

Results, Conclusions, and Discussions:: Using Transmission Electron Microscopy (TEM), we show that particle shape shifts from a typical cubic shape (PBNP) to a more spherical appearance (Exo:PB). Further electron mapping result using uranyl acetate staining for exosome identification was used to validate successful exosome coating on a PBNP core for the Exo:PB particles (Figure 1A.). Exosome markers were validated with western blot analysis, and cellular uptake patterns were observed using U-87 cells to validate biological similarities to native exosomes. Photo-conversion abilities of Exo:PB were seen on the in vitro level through live/dead cell staining and MTT assays when exposed to an 808 nm laser (Figure 1B.). Finally, particles were injected into both subcutaneous and orthotopic glioblastoma mouse models to observe therapeutic effects and targeting efficiency. Exo:PB was shown to have a great photothermal effect with entire ablation of the tumor region in the subcutaneous model compared to the PBS control (Figure 1C.). Once intravenously injected into the orthotopic model, the particles showed great uptake within the tumor region with high specificity, as validated through photoacoustic imaging, H&E staining and overlay fluorescence (Figure 1D.).

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