Abstract
Purpose :
Retinoblastoma (Rb), the most common childhood ocular cancer, often results from RB1 genetic mutation. Upregulation of the neuroblastoma oncogene MYCN is linked to 11% of pediatric cancers and contributes to Rb. Major drawbacks with the current treatments include invasive and repeated dosing, which can cause endophthalmitis, retinal detachment, and vitreous hemorrhage. Moreover, pharmaceutical compounds at higher doses may cause systemic side effects. Drug-loaded Extracellular vesicles (EVs) can be used as a drug delivery system to overcome these limitations by delivering a dose designed to specifically target RB cells.
Methods :
Different concentrations of doxorubicin hydrochloride (Dox) were loaded on Y79 cells. Cell conditioned media was collected from Dox-loaded Y79 cells and Dox-loaded EVs were isolated successfully via ultracentrifugation. The biophysical properties of these EVs were assessed using nanoparticle tracking analysis (NTA), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and ZetaSizer. To visualize EVs, 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate (DiI) fluorescent dye was employed for labeling, enabling the characterization of nanoparticle cellular uptake through fluorescence microscopy. Flow Cytometry and Western blot analysis were performed to elucidate the presence of CD63 and CD81 (EV markers). The cytotoxicity of EVs of 2.5,5, and 10µg/mL Dox or Dox-loaded EVs against Y79 cells was evaluated by Presto Blue analysis.
Results :
The biophysical characteristics of drug loaded EVs showed a spherical morphology via TEM; with their particle size of 110.7 ± 20 nm and particle concentration of 2 x 108 particles/mL via Nanosight. The EVs showed a surface charge of 0.5 ± 0.8 mV via DLS. Dil-labelled EVs were successfully up taken by the Y79 cells as visualized under fluorescence microscopy. Western blot and flow cytometry results confirmed the presence of CD63 and CD81in drug loaded EVs. Higher concentrated dox-loaded EVs showed significant cytotoxicity against Y79 cells in comparison to free dox treatment (p value-** p < 0.01).
Conclusions :
The findings of this study offer valuable insights into the potential of EV drug delivery to enhance treatment efficacy and mitigate adverse effects related to current Rb treatment.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.