Preparation of chitosan nanoparticle containing recombinant CD44v antigen and evaluation of its immunization capacity against breast cancer in BALB/c mice

Abstract

Objective(s): Breast tumors show heterogeneity containing cancer stem cells as a small subpopulation of a tumor mass. CD44 as a cancer stem cells antigen is abnormally expressed by carcinomas of epithelial origin. Also, overexpression of CD44 variable isoforms (CD44v) is associated with malignancy in breast cancer. In the present research, our objective was to evaluate the immunogenicity of prepared nanoparticles containing a novel recombinant CD44v (rCD44v) protein in the mouse model. Materials and methods: CD44 gene was expressed in E. coli BL21 DE3 using the pET28a-CD44 vector. The expressed rCD44v protein was purified, encapsulated into the chitosan nanoparticles, and administered to BALB/c mice. ELISA was used to evaluate the immunoglobulin levels of immunized animals. For challenge experiment, 2 × 106 4T1-CD44 tumor cells were injected subcutaneously in mice, and tumor size, necrosis, and metastases were measured. Finally, cell proliferation assay, cytokines assay, and neutralization assay of the mouse anti-rCD44v on the human breast cancer cell line were examined. Results: The measured size of chitosan-rCD44v nanoparticles was 146.5 nm. Recombinant CD44v encapsulated by chitosan nanoparticles increases immunological responses via the adjuvant nature of chitosan nanoparticles. In the immunized mice, IgG and IgA titers were significantly increased. Tumor growth in injection and nano-injection test groups compared with the mice control groups displayed a significant reduction (P < 0.05). A high amount of splenocytes secreting IFNγ and IL-17 was seen in immunized mice with rCD44v (P < 0.05). Furthermore, a smaller size of lung metastases compared to the control mice groups was detected. Conclusion: The encapsulated rCD44v within the chitosan nanoparticles induced a significant immune response in mice and can establish significant protection against breast cancer. Therefore, it can be considered a vaccine candidate for breast cancer therapeutic modalities.