Preparation and evaluation of norcantharidin-encapsulated liposomes modified with a novel CD19 monoclonal antibody 2E8

Abstract

In this study, norcanthridin (NCTD)-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 (2E8-NCTD-liposomes) and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19⁺ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody (mAb). NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19⁺ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19⁺ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG₂₀₀₀-DSPE reached 1:50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19⁺ leukemia cells was significantly higher than that on CD19-leukemia cells. Similarly, the targeting efficiency of the immunoliposomes was also higher than that of the NCTD-liposomes on CD19⁺ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nalm-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.

Keywords

targeting / immunoliposome / 2E8(CD19) / norcantharidin

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