Cisplatin encapsulated human macrophage-engineered vesicles for utilization in ovarian cancer treatment

David Schweer; Namrata Anand; Connie Cao; J. Robert McCorkle; Frederick Ueland; Christopher Richards; Jill Kolesar

doi:10.1016/j.ygyno.2024.07.282

Objectives Macrophage-derived vesicles have cancer-targeting properties that allow for the selective delivery of chemotherapy directly to cancer cells. The purpose of this study was to compare the inhibition of cell proliferation and double-stranded (ds) DNA damage between cisplatin-encapsulated human macrophage-engineered vesicles (cisMEVs) and free cisplatin in ovarian cancer cell lines. Methods Ovarian adenocarcinoma cell lines CAOV3 and OVCAR3 were cultured in appropriate media. Macrophages were isolated and cultured from human peripheral blood mononuclear cells obtained from a local blood center. Macrophages were stimulated to a M1 phenotype using LPS/IFN-γ. CisMEVs were generated from M1 macrophages with nitrogen cavitation within an 8.33 mM cisplatin solution and ultracentrifugation. Ovarian cancer cells were plated at 5 x 103 cells/well in 96-well plates and incubated at 37˚C for 24 h prior to treatment. CisMEVs and free drug concentrations were prepared using serial dilutions. CisMEV cisplatin concentrations were measured with liquid chromatography-mass spectrometry (LC/MS). A cell viability assay was performed 72 h following the addition of cisplatin or cisMEVs to CAOV3 and OVCAR3 cells MEVs according to the manufacturer’s instructions (CellTiter-Glo 2.0, Promega). Separate plates were analyzed after 24 h for DNA damage using a γ-H2AX immunofluorescence assay and a CellInsight CX7 High Content Analysis Platform according to the manufacturer’s instructions (Thermo Scientific). DNA damage was quantified as fold change in γ-H2AX signal intensity relative to vehicle control. Dose-response curves were calculated using GraphPad Prism 5. CisMEVs and MEVs were compared using one-tailed paired t-tests or unpaired t-tests for IC50 and fold changes, respectively. Experiments were performed in triplicate. Results Compared to free cisplatin, cisMEVs demonstrated statistically significant enhanced inhibition of proliferation in CAOV3 (IC50: 1.413 ± 0.6505 vs 5.946 ± 2.156 μM; P = 0.0447) and approached significance in OVCAR3 (2.312 ± 0.4930 vs 3.819 ± 1.285; P = 0.0772). In regard to dsDNA breaks utilizing γ-H2AX staining levels, when comparing cisMEV concentrations of 3.9 μM (average dose retrospectively determined via LC/MS) to free cisplatin concentrations of 5 μM, cisMEVs demonstrated statistically significant differences in CAOV3 (Mean fold change: 2.812 ± 0.3154 vs 1.876 ± 0.06574; P = 0.0012) and OVCAR3 cells (Mean fold change: 10.34 ± 2.613 vs 4.386 ± 0.4013; P = 0.0046). Conclusions CisMEVs display greater proliferation inhibition and increased dsDNA breaks compared to free cisplatin in ovarian cancer cells in vitro.

Cisplatin encapsulated human macrophage-engineered vesicles for utilization in ovarian cancer treatment

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