Functional characterization of a macrophage-polarizing agent loaded in reconstituted high-density lipoprotein nanoparticles for cancer immunotherapy

Purpose: The murine stimulator of interferon genes agonist 5,6-dimethylxanthenone-4-acetic acid (DMXAA) has been successfully used to modulate the function of macrophages from an immunosuppressive phenotype (M2) to a proinflammatory phenotype (M1). Drugs, such as DMXAA, that facilitate this phenotype reversal are particularly relevant in cancer therapy as the immunosuppressive phenotype of tumor-associated macrophages (TAMs) contributes to tumor progression. This functional reprogramming of TAMs by drugs, including DMXAA, has been reported to facilitate sustained tumor regression. However, drug targeting strategies are needed to specifically deliver reversal agents to TAMs and avoid off-target effects. In this study, we investigated the physicochemical characteristics, as well as phenotype reversal capability of DMXAA encapsulated in the biocompatible reconstituted high-density lipoprotein (rHDL) nanoparticles. Methods: The DMXAA-loaded rHDL (rHDL-DMXAA) was prepared using an enhanced microfluidics apparatus. Dynamic light scattering (DLS) and fast protein liquid chromatography (FPLC) were used to assess particle size and preparation homogeneity. M1 and M2 markers expression by murine macrophages were assessed via western blot and ELISA. Results: While DLS indicated particles of 33.9 ± 14 nm diameter size, the FPLC profile of preparation indicated a homogeneous preparation with an estimated particle molecular weight of 132 kDa. Upon treatment with rHDL-DMXAA, the macrophages showed increased M1 marker expression. Conclusion: rHDL-DMXAA induced an M1 phenotype in macrophages and its characteristics suggest a potential for intratumoral accumulation. Thus, the rHDL-DMXAA represents a functional proof-of-concept strategy to target macrophages with implications in cancer immunotherapy.