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Doxorubicin-loaded NK exosomes enable cytotoxicity against triple-negative breast cancer spheroids
Zahra Sadat Hashemi1, Mahlegha Ghavami2, Fateme Mohammadi3
1ATMP Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran.
Objectives:
Natural killer (NK) cells are the most professional innate immune cells that initiate extracellular apoptosis via cytotoxic granules in malignant cells. Antitumoral properties of NK-derived exosomes (Exos) are attributed to their parent cells. Loading drugs into Exos as a carrier can enhance their effect and enable targeted delivery. In the present study, we aim to deliver Doxorubicin (DOX) to the breast cancer spheroids by NK-Exos.
Materials And Methods:
Peripheral blood mononuclear cells (PBMC) were used to harvest NK cells, and NK-Exos were isolated from NK cell expansion medium using an Exo-spinTM kit. DOX was loaded via the ultrasonication method. AO/EtBr, Annexin/PI, DAPI, MTT, and spheroids of human breast cancer were used to track the cytotoxic effect of DOX-NK-Exos. The colony formation assay, scratch and transwell assays, Real-Time PCR for p53 and VEGF-A, and WB for protein expression were also performed.
Results:
When compared to free DOX, all viability tests validated the inhibitory effects of DOX-NK-Exos. The obtained results indicated that DOX-NK-Exos selectively reduced tumor cell viability and spared fibroblast and MCF-10A as noncancerous cells. Long after spheroid treatment, DOX-NK-Exos' remarkable effect persisted.
Conclusion:
Human breast carcinoma mass treated with DOX-NK-Exos underwent apoptosis and showed a strong inhibitory effect on proliferation. Thus, they can reduce the side effects of chemotherapeutics and can be used as drug carriers with selective toxicity. Additionally, the additive action of this combination formula results in a more severe loss in cell viability.
Insights
Natural killer cell-derived exosomes loaded with Doxorubicin selectively kill breast cancer cells, sparing healthy cells. This targeted delivery reduces chemotherapy side effects and enhances anti-tumor efficacy.
Area of Science:
- Immunology
- Nanotechnology
- Oncology
Background:
- Natural killer (NK) cells are crucial innate immune cells targeting malignant cells via cytotoxic granules.
- NK-derived exosomes (Exos) possess intrinsic anti-tumor properties, making them promising for drug delivery.
- Exosomes can be engineered as carriers for targeted drug delivery, enhancing therapeutic efficacy and minimizing side effects.
Purpose of the Study:
- To investigate the potential of Doxorubicin (DOX)-loaded NK-Exos for targeted delivery to breast cancer spheroids.
- To evaluate the selective cytotoxic effects of DOX-NK-Exos on breast cancer cells compared to non-cancerous cells.
- To assess the anti-proliferative and apoptosis-inducing capabilities of DOX-NK-Exos in human breast carcinoma models.
Main Methods:
- NK cells were isolated from peripheral blood mononuclear cells (PBMC).
- NK-Exos were purified and loaded with Doxorubicin (DOX) using ultrasonication.
- Cytotoxicity was assessed using viability assays (MTT, AO/EtBr, Annexin/PI, DAPI) on breast cancer spheroids and non-cancerous cells.
- Proliferation and invasion were evaluated using colony formation, scratch, and transwell assays. Gene and protein expression (p53, VEGF-A) were analyzed via Real-Time PCR and Western Blot.
Main Results:
- DOX-NK-Exos demonstrated significant tumor cell viability inhibition compared to free DOX.
- DOX-NK-Exos exhibited selective toxicity, effectively targeting breast cancer cells while sparing fibroblasts and MCF-10A cells.
- Sustained therapeutic effects were observed long after spheroid treatment with DOX-NK-Exos.
Conclusions:
- DOX-NK-Exos induce apoptosis and inhibit proliferation in human breast carcinoma, confirming their anti-tumor potential.
- Engineered NK-Exos serve as effective drug carriers with selective toxicity, potentially reducing chemotherapy side effects.
- The combination of DOX and NK-Exos exhibits an additive effect, leading to a more pronounced loss of cancer cell viability.
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