Imaging and Treatment of Primary Tumors and Metastases by Targeted Macrophage Therapy, Using Radiation Therapy-Directed Encapsulated Nanoparticles

Abstract

Liposome-protamine-hyaluronic acid nanoparticles (LPH-NPs; 173 ± 7.2 nm in diameter) encapsulated in oxygen-rich microcapsules (3.6 ± 0.8 μm in diameter) that release their contents upon exposure to radiation were used in two radiotherapy sessions. We investigated primary tumor and metastasis imaging and macrophage-based therapy through silencing of “don’t eat me” signals and an increase in tumor-associated macrophages (TAMs) of M1 phenotype. For the first radiotherapy session, LPH-NPs, containing 5% iopamiron, and 250 nmol anti-CD47 small interfering RNA (siRNA) were mixed with 1.0 mL oxygen-rich solution (2200 ppm O2) containing 4.0% alginate, 3.0% hyaluronate, 1 mg ascorbate, and 1 μg/mL CD-40 and sprayed into 0.5 mmol/L FeCl2 supplemented with 1 μg/mL anti-vascular endothelial growth factor receptor (VEGFR)-1/2 antibody (Ab). The resultant microcapsules (1 × 1010) were intravenously (i.v.) administered into C57BL/6 mice with a primary MC38 tumor in the left hind leg and lung metastases, and their accumulation in the tumor was monitored by computed tomography (CT). Subsequently, the primary tumors and metastases were treated with 10 or 20 Gy 60Co γ-radiation. For the second session, microcapsules were generated by mixing LPH-NPs containing 200 μg anti-CD40 monoclonal Ab (moAb) and 900,000 IU interleukin (IL)-2, with the same oxygen-rich cocktail used in session one, and were sprayed into 0.5 mmol/L FeCl2 supplemented with 1 μg/mL anti-P-selectin Ab. They were then i.v. administered into the mice and allowed to interact with P-selectin for 9 h prior to a second radiotherapy with 10 or 20 Gy 60Co γ-radiation. In session one, the anti-VEGFR-1/2 microcapsules accumulated around the primary and metastatic tumors as detected by CT, and in response to the irradiation, they released anti-CD 47 siRNA LPH-NPs and oxygen-rich water, which silenced “don’t eat me” signals and increased tumor oxygen concentration, respectively. In session two, the microcapsules accumulated around the primary tumor through a P-selectin antigen-antibody reaction, and in response to the irradiation, they released anti-CD 40 moAb/IL-2 LPH-NPs and oxygen-rich water. Additionally, the radiation recruited TAMs in the tumors. The released anti-CD 40 moAb/IL-2 switched the phenotype of the recruited TAMs from M2 to M1, and the increased oxygen concentration due to the repeatedly released oxygen-rich water inhibited TAM repolarization from M1 to M2, leading to an increase in TAMs of M1 phenotype, which then attacked the tumors for which the “don’t eat me” signals were already silenced in session one. These treatments significantly increased the antitumor effect (EF 1.8) and reduced metastasis by 82.6%. Our CT-detectable microcapsules exhibited targeted macrophage therapy, improving tumor diagnosis and treatment.