Macrophages play a critical role in the immune response due to their ability to recognize and remove pathogens, as well as present antigens, which are involved in inflammation, but they are also one of the most abundant immune cell populations present in the tumor microenvironment. In recent years, macrophages have become promising cellular carriers for drug and nanoparticle delivery to the tumor microenvironment, mainly due to their natural properties such as biocompatibility, degradability, lack of immunogenicity, long half-life in circulation, crossing biological barriers, and the possibility of migration and accumulation at a site of inflammation such as a tumor. For the effectiveness of this therapeutic strategy, known as “Trojan horse”, it is important that the nanoparticles engulfed by macrophages do not affect their proper functioning. In our review, we discussed how the size, shape, chemical and mechanical properties of nanoparticles influence their internalization by macrophages. In addition, we described the promising research utilizing macrophages, their cell membranes and macrophage-derived exosomes as drug carriers in anticancer therapy. As a prospect of the wider use of this therapeutic strategy, we postulate its future application in boron delivery to the tumor environment in boron neutron capture therapy.

Boron carbide is one of the hardest materials in the world which can be synthesized by various methods. The most common one is a carbothermic or magnesiothermic reduction of B₂O₃ performed at high temperatures, where the obtained powder still requires grinding and purification. The goal of this research is to present the possibility of synthesizing B₄C nanoparticles from elements via vapor deposition and modifying the morphology of the obtained powders, particularly those synthesized at high temperatures. B₄C nanoparticles were synthesized in the process of direct synthesis from boron and carbon powders heated at the temperature of 1650 °C for 2 h under argon and characterized by using scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction analysis, and dynamic light scattering measurements. The physicochemical characteristics of B₄C nanoparticles were determined, including the diffusion coefficients, hydrodynamic diameter, electrophoretic mobilities, and zeta potentials. An evaluation of the obtained B₄C nanoparticles was performed on several human and mouse cell lines, showing the relation between the cytotoxicity effect and the size of the synthesized nanoparticles. Assessing the suitability of the synthesized B₄C for further modifications in terms of its applicability in boron neutron capture therapy was the overarching goal of this research.

Background: The use of cells as carriers for the delivery of nanoparticles is a promising approach in anticancer therapy, mainly due to their natural properties, such as biocompatibility and non-immunogenicity. Cellular carriers prevent the rapid degradation of nanoparticles, improve their distribution, reduce cytotoxicity and ensure selective delivery to the tumor microenvironment. Therefore, we propose the use of phagocytic cells as boron carbide nanoparticle carriers for boron delivery to the tumor microenvironment in boron neutron capture therapy. Results: Macrophages originating from cell lines and bone marrow showed a greater ability to interact with boron carbide (B₄C) than dendritic cells, especially the preparation containing larger nanoparticles (B₄C 2). Consequently, B₄C 2 caused greater toxicity and induced the secretion of pro-inflammatory cytokines by these cells. However, migration assays demonstrated that macrophages loaded with B₄C 1 migrated more efficiently than with B₄C 2. Therefore, smaller nanoparticles (B₄C 1) with lower toxicity but similar ability to activate macrophages proved to be more attractive. Conclusions: Macrophages could be promising cellular carriers for boron carbide nanoparticle delivery, especially B₄C 1 to the tumor microenvironment and thus prospective use in boron neutron capture therapy. Graphical

Introduction: Boron neutron capture therapy (BNCT) is a promising targeted radiotherapy that enables the treatment of cancers at the cellular level. The crucial aspect of BNCT are boron carriers, which should selectively reach cancer cells by delivering high concentrations of boron. Therefore, we propose the use of boron carbide (B₄C) nanoparticles functionalized with antibodies directed against receptors overexpressed in cancer cells, such as the low-density lipoprotein receptor (LDLR) and the epidermal growth factor receptor (EGFR). Methods: Hydrodynamic diameter measurements confirmed the stability of functionalized B₄C nanoparticles in culture media during biological tests lasting up to 72 hours. The toxicity of the nanoparticles was assessed using the MTT assay and BrdU cell cycle assay on three types of cancer cells (PC-3, T98G, and SCC-25) with different levels of LDLR and EGFR surface expression. The uptake of functionalized B₄C nanoparticles by cancer cells was assessed based on flow cytometry, fluorescence microscopy, and holotomography. Boron concentrations in cancer cells were quantified via ICP-MS. Results: Functionalized B₄C nanoparticles showed even 2-fold higher interaction with SCC-25 cells characterized by the highest surface expression of both receptors than with PC-3 and T98G cells. Holotomographic imaging confirmed the greater intracellular uptake of functionalized B₄C nanoparticles compared to unmodified B₄C, providing further evidence for the selective targeting of boron to cancer cells. ICP-MS analyses showed that B₄C anti-LDLR nanoparticles were the most effective in delivering a high boron concentration to cancer cells. Particularly in SCC-25 cells, the concentration was 9.58 ± 2.6 mg/L boron per million cells. The highest uptake by these cells was associated with a decrease in viability to 63% and a slight reduction in the percentage of cells in S phase after 24-hour exposure. Conclusion: Stable complexes of antibody-functionalized B₄C nanoparticles were successfully obtained, demonstrating increased tropism towards cancer cells overexpressing LDLR and EGFR.

In this research, we developed boron-rich nanoparticles that can be used for boron neutron capture therapy as potential carriers for boron delivery to cancerous tissues. Functionalized carbonated boron nitride nanostructures (CBNs) were successfully synthesized in self-propagating combustion waves in mixtures of high-nitrogen explosives and boron compounds. The products’ composition, morphology, and structural features were investigated using Fourier transform infrared spectroscopy, powder X-ray diffraction, low-temperature nitrogen sorption analysis, thermogravimetric analysis, high-resolution scanning electron microscopy, and high-resolution transmission electron microscopy. The extreme conditions prevailing in combustion waves favor the formation of nanosized CBN hollow grains with highly disordered structures that are properly functionalized on the surface and inside the particles. Therefore, they are characterized by high porosity and good dispersibility in water, which are necessary for medical applications. During biological tests, a concentration-dependent effect of the obtained boron nitride preparations on the viability of normal and neoplastic cells was demonstrated. Moreover, the assessment of the degree of binding of fluorescently labeled nanoparticles to selected cells confirmed the relationships between the cell types and the concentration of the preparation at different incubation time points.

Introduction: Boron neutron capture therapy (BNCT) is a targeted radiotherapy that represents a promising treatment for glioblastoma multiforme. BNCT efficacy depends on selective boron carriers capable of crossing the blood–brain barrier (BBB). Therefore, we propose an original strategy for boron delivery in BNCT, based on the cellular carriers. This study assessed the ability of bone marrow-derived macrophages (BMDMs) to accumulate boron carbide (B₄C) nanoparticles, transport them across an in vitro BBB model toward glioblastoma cell-conditioned medium, and interact with tumor cells. Methods: The uptake and accumulation of B₄C nanoparticles by BMDMs in different polarization states (M0, M1, M2) were confirmed by holotomography and ICP-MS. The scratch assay assessed spontaneous migration of BMDMs loaded with B₄C nanoparticles. The Transwell system was used to determine the ability of BMDMs with B₄C to cross bEnd.3 brain endothelial cell monolayer, mimicking the BBB, toward GL-261 glioblastoma cell-conditioned medium rich in CCL2. Macrophage interactions with glioblastoma spheroids were investigated using the CellTiter-Glo 3D Cell Viability Assay and flow cytometry. Results: All BMDM populations demonstrated the ability to accumulate B₄C nanoparticles, with the significantly highest boron concentration detected in M1 macrophages (21.53 ± 1.64 mg/L per 10⁶ cells). Moreover, M0, M1, and M2 macrophages loaded with B₄C nanoparticles were capable of migrating through the brain endothelial cell monolayer, toward the glioblastoma cell-conditioned medium. BMDMs with nanoparticles did not affect the viability of glioblastoma spheroids after 6 days of co-culture, in contrast to macrophages without nanoparticles, which increased the survival of tumor cells. Importantly, CD206 expression was not increased in M1 macrophages after contact with spheroids, and was lower in M2 macrophages loaded with B₄C nanoparticles compared to control M2 macrophages. Conclusion: BMDMs are promising carriers of B₄C nanoparticles for BNCT due to their ability to cross the in vitro BBB model toward the glioblastoma microenvironment. The infographic illustrates the process involving bone marrow-derived macrophages (BMDMs). It begins with the preparation of BMDMs from C57BL/6 mice, isolating bone marrow cells from femur and tibia and differentiating them into M0, M1 and M2 macrophages. These BMDMs are then incubated with boron carbide nanoparticles. The next step shows the co-culture of BMDMs with glioblastoma spheroids. The analysis of boron carbide effects on BMDMs includes flow cytometry for BMDM markers, MTT viability assay, visualization of uptake using holography and boron concentration analysis by ICP-MS. The migration ability of BMDMs loaded with boron carbide is assessed through a scratch assay and a Transwell assay, showing migration across a blood-brain barrier model. Finally, BMDM and spheroid interactions are analyzed using a CellTiter-Glo 3D cell viability assay, CCL2 production via ELISA assay and flow cytometry for BMDM markers.BMDMs prep, boron carbide incubation, migration and glioblastoma spheroid interaction infographic.