Introduction:: After over a century of development, cancer immunotherapy(CIT) has risen to become a promising approach for treating cancer. Unlike conventional methods that try to kill cancer cells directly, CIT involves modifying or stimulating the immune system's natural ability to fight the disease. So far, various CIT treatments have shown remarkable results in terms of survival rates, and many new and combination strategies are currently under study. Despite their undeniable potential, CIT still has many hurdles to overcome. Tumors are complex, adaptive, and heterogeneous, and the tumor immune microenvironment, called TIME, has many internal and external influencing factors that remain poorly understood. Recently, an antitumor strategies using nanotechnologies to favorably change the immunosuppressive nature of the TIME have been introduced. They can change the immune-defective environments into immunogenic and immunoreactive environments. Stimulator of Interferon Genes (STING) activation has been investigated as a co-stimulatory strategy to boost the immune response against cancer by mediating interferon type I responses in both innate and adaptive immune systems and by activating cell death. Targeted delivery of STING agonists could potentially activate immune-suppressed tumors, turning them from cold to hot. Macrophages accumulate in tumors by proliferation from tissue resident precursors or by trafficking from bone-marrow-derived precursors. Once in tumors, these cells can adopt a tumor-promoting phenotype that induces immunosuppression, angiogenesis, tumor growth, and metastasis. The presence of TAMs are releated to immunosuppression, but M1 are related immunostiumulation and T cell activation. Therefore, the replorization from TAM to M1 is one of the ways to reprogram TIME.
Materials and Methods:: Monochloroacetic acid was purchased from Sigma Aldrich.
< Synthesis of carboxymethyl guar gum, CMGG > Guar gum (GG) and sodium hydroxide was stirred for 30 min. After 30 min, monochloroacetic acid was added on the solution and stirred for 50 min at 70°C. < Modification of CMGG > - Polymer 1 (Guanidinylated GG, GGG) CMGG, ethylenediamine and 1H-pyrazole-1-carboxamidine hydrochloride were used.
- Polymer 2 (Guanidinylated and imidazoled GG, GIGG) CMGG, ethylenediamine, 1H-pyrazole-1-carboxamidine hydrochloride and 1-(3-Aminopropyl)imidazole were used.
- Polymer 3 (STING-activating GG, STGG)
CMGG, ethylenediamine, 1H-pyrazole-1-carboxamidine hydrochloride and hexamethyleneimine were used.
< Synthesis of MnO2 NPs > Sodium thiosulfate and potassium manganate (VII) solution were stirred for 1hr.
< Coating MnO2 NPs with polymers > Polymers were added into the MnO2 solution by dropwise for 18hr.
< Bone marrow derived macrophage (BMDM) harvesting > 8 weeks old C57BL/6N mice were sacrificed. Mouse M-CSF (JW creagene) was used to polarize to the macrophage.
< Polarization to M1 and M2 > Interferon gamma and IL-4 were used for polarization of M1 and M2.
< Cell cytotoxicity test > M1 and M2 type macrophage were seeded on 96 well plate and were treated on next day. After 24hr, cell viability were measured using MTT assay.
< Cellular uptake > Cells were seeded on 24 well plate and were treated. Cells were harvested and analyzed from flow cytometry.
< Analysis of gene expression > Expression of gene were analyzed from RT-PCR
Results, Conclusions, and Discussions:: 1H NMR data showed the successful modification of guar gum (GG). Each polymers was modified from carboxymethyl guar gum (CMGG) (Fig 1). Each polymers showed low cytotoxicity for M1 type and M2 type macrophages (Fig 2). 2hr was sufficient for uptake of polymers to M2-type macrophage and they showed the higher cellular uptake compared to M1-type macrophage (Fig 3). Manganese dioxide with polymers showed the successful reprogramming from M1-type macrophage. Expressions of Nos2, Cd86, Il6 and Tnfa genes were increased, whereas expressions of Arg1, Mrc1 and Chi3l3 genes were decreased (Fig 4). Three polymers synthesized were classifed by the potential of endosomal escape and STING activation. All three polymers included the guanidine moiety to coat the surface of the manganese dioxide nanoparticle, called MnO2 NP using electrostatic interaction. Second polymer included the imidazole moiety to escapae the endosome by inducing the proton sponge effect. Third polymer included the azepane moiety to escape the endosome and to activate the STING pathway. Fig 1. showed different intensity of peaks on 1H NMR. Different ratio of each moieties in each polymers were conjugated to find the highest tumor-associated macrophage (TAM) reprogramming ability. For successful reprogramming, the macrophage should not be depleted, so the polymers should not influence on the viability of macrophages as Fig 2. The presence of mannose backbone made the polymers bind the mannose receptors. All polymers had the mannose backbone, so they could target the mannose receptors of M2-type macrophage successfully. Fig 3. showed the selectivity of cellular uptake between M1-type and M2-type macrophage.MnO2 NP was degraded as manganese ion to activate cGAS-STING pathway. cGAS-STING pathway could be involved in the reprogramming process as activating the production of pro-inflammatory cytokines. Therefore, MnO2 NP with polymers could target and reprogram the M2-type macrophage as Fig 4.
