Ovarian cancer remains among the ten most diagnosed malignancies globally, with a high mortality rate. In 2020, over 200,000 deaths were attributed to this malignancy worldwide. Because ovarian cancer frequently progresses to late stages before diagnosis, it has a low survival rate, below 50%.1
Conventionally, ovarian cancer treatment involves surgery followed by chemotherapy with taxanes and platinum-based drugs.2 Most patients, ~80%, show complete responses to this first treatment course and remain disease-free for over one year. However, disease recurrence is frequent, especially for those with advanced disease, necessitating a second course of chemotherapy. At this stage, drug resistance and toxicities are major challenges for re-implementing chemotherapy.1,2
Paclitaxel is a plant-derived taxane frequently used as a chemotherapeutic agent in ovarian cancer.3 Despite being an effective therapy for various cancer indications, its generalized mechanism of action (i.e., microtubule stabilization, mitotic arrest, and cell proliferation inhibition) results in off-target toxicities following systemic administration. Several strategies have been implemented to enable a more tumor-targeted effect, including its conjugation to small molecules, antibodies, or aptamers and the encapsulation of Paclitaxel in nanoparticles.4
Advantages of nanoparticles in chemotherapy delivery
The use of nanoparticles for the delivery of Paclitaxel and other chemotherapy drugs provides several benefits, including the potential for specific tumor targeting, controlled release, and improved stability. Stable protein-based synthetic nanoparticles encapsulating Paclitaxel proved effective in a glioma-bearing mouse model, extending survival and reducing tumor size while preventing liver toxicity.5
Naturally derived vesicles or extracellular vesicles, such as exosomes, can also be leveraged in chemotherapy drug delivery. Extracellular vesicle nanoparticles offer various benefits as delivery vehicles, including low toxicity and immunogenicity and the capacity to traverse physiological barriers of interest, such as the blood-brain barrier. Previously, exosomes derived from brain endothelial cells were shown to serve as an efficient vehicle to deliver chemotherapy drugs in a zebrafish brain cancer model, leading to tumor cytotoxicity.6
Engineered extracellular vesicles for ovarian cancer
Beyond their favorable physiological properties, extracellular vesicles can be modified further to make them even more useful for cancer drug delivery. In new work led by Professor Carlos Salomon Gallo at the Centre for Clinical Research, University of Queensland, extracellular vesicles have been engineered to achieve improved cargo delivery to ovarian cancer cells.2
In their new publication, Alharbi and colleagues have engineered extracellular vesicles to present the ligand for the ephrin-B4 receptor, ephrin-B2, on the surface. Briefly, extracellular vesicles were prepared from HEK293T cells transfected with a plasmid encoding LAMP2b fused to GFP and ephrin-B2. Once expressed, the chimeric membrane-bound LAMP2b protein supported intra- and extra-vesicular GFP and Ephrin-B2 expression, respectively.
Extracellular vesicles were isolated from the cell culture supernatant through differential centrifugation and size exclusion chromatography, followed by various analyses to validate their expected properties, such as their size and expression of various markers (e.g., CD63, CD81, and CD9). Additionally, the expression of ephrin-B2 was confirmed via MS/MS analysis.
“Two expression vectors were constructed: a control vector that expresses LAMP2b with a C-terminally fused enhanced green fluorescent protein (EGFP; hereafter, simply referred to as GFP), and one that expresses LAMP2b with a C-terminally fused GFP and an N-terminally fused ephrin-B2 (EFNB2) peptide for targeting the ephrin-B4 receptor. Each construct (LAMP2b-GFP and EFNB2-LAMP2b-GFP) was cloned into a pCMV-3 Tag-1A-P2A expression vector backbone, downstream of the CMV promoter between NotI and ApaI restriction sites (Vectors were designed in house and generated by Genscript).”2Retrieved from Alharbi et al. 2024, only a portion of the abstract figure is shown. Deed - Attribution 4.0 International - Creative Commons
in vitro and in vivo uptake of extracellular vesicles
By decorating the surface of extracellular vesicles with ephrin-B2, the team hoped to improve their uptake via receptor-mediated endocytosis by ovarian cancer cells, which overexpress the ephrin-B4 receptor. Internalization of extracellular vesicles expressing ephrin-B2-LAMP2b-GFP and those only expressing the control LAMP2b-GFP was evaluated first in vitro, using cultured ID8 mouse ovarian cancer cells. The team found that both vesicles were similarly internalized by ID8 cells in culture. In contrast, following intravenous injection of extracellular vesicles in an ID8 ovarian tumor-bearing mouse model, the internalization of ephrin-B2-LAMP2b-GFP extracellular vesicles was significantly higher than the control within the tumor.
Not surprisingly both types of extracellular vesicles were also internalized to a similar extent in various organs (e.g., spleen, brain, and lung). Additionally, as is the case with many different delivery vehicles, including lipid nanoparticles (LNPs), the highest internalization of extracellular vesicles occurred in the liver.
Conclusions
In their study, the University of Queensland team investigated the potential of extracellular vesicles with surface expression of ephrin-B2 for targeting ovarian cancer cells. Their in vivo findings suggest that these engineered extracellular vesicles could offer a targeting advantage. However, the authors note that using extracellular vesicles for delivering therapies is a very complex process. One significant challenge is the absence of standardized workflows based on clinical regulatory guidance for large-scale production of optimized extracellular vesicles.
Reference
1. Tavares, V. et al.,(2024) A Comprehensive Review of Ovarian Cancer Management in an Era of Advancements. Int. J. Mol. Sci. https://doi.org/10.3390/ijms25031845
2. Alharbi, M. et al., (2024) Enhancing precision targeting of ovarian cancer tumor cells in vivo through extracellular vesicle engineering. International Journal of Cancer. https://doi.org/10.1002/ijc.35055
3. Ying, N. et al., (2023). Nano delivery system for paclitaxel: Recent advances in cancer theranostics. Colloids and Surfaces B: Biointerfaces, https://doi.org/10.1016/j.colsurfb.2023.113419
5. Mauser, A. et al., (2024) Controlled Delivery of Paclitaxel via Stable Synthetic Protein Nanoparticles. Advanced Therapeutics, 2400208. https://doi.org/10.1002/adtp.202400208
6. Yang, T. et al., (2015) Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm Res. https://doi.org/10.1007%2Fs11095-014-1593-y
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