(T0930-02-08) The Influence of Silica Nanoparticles with Varied Physicochemical Properties on the Immune Function of RAW 264.7 Macrophages

Purpose: Silica nanoparticles (SNPs) have been explored for their potential in controlled drug delivery and imaging applications (1–3). However, there is no FDA-approved drug that uses SNPs as an injectable delivery system. One of the reasons hindering the clinical translation of SNPs is limited knowledge about their potential toxicity to immune cells. Variations in the physicochemical properties of SNPs can have an impact on their potential immunotoxicity upon systemic delivery. In this study, we synthesized SNPs with variations in physicochemical properties, established the maximum nontoxic dose and time to saturate RAW 264.7 murine macrophages with no visible toxicity, and investigated functional changes of these cells.

Methods: Nonporous Stöber SNPs, mesoporous, and hollow mesoporous SNPs were synthesized and characterized with transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Fourier Transform Infrared Spectroscopy (FTIR). Particle solutions were tested for endotoxin content. RAW 264.7 cells were cultured, and a CCK-8 cell viability assay was performed to determine the maximum nontoxic concentration of the SNPs in the cells. Ion-coupled plasma mass spectroscopy (ICP-MS) was performed to determine the Silicon (Si) content inside the cells at different time points and the maximum internalized SNPs. Live/dead assay was performed with annexin/PI and analyzed with flow cytometry to observe the live, apoptotic, and necrotic cell population upon SNP internalization. CD80 surface marker level was determined to establish the M1/M2 phenotypic changes of the macrophages upon SNP treatment using flow cytometry. Membrane integrity changes were studied using lactate dehydrogenase (LDH) assay, and a red zymosan-based phagocytosis assay was performed to analyze the effect on the phagocytic activity of macrophages.

Results: Nonporous Stöber SNPs with an average diameter of 50 nm (SNP50) and 100 nm (SNP100), and mesoporous (MSNP) and hollow mesoporous SNPs (HMSNP) with average diameters of around 100 nm were synthesized. The maximum nontoxic doses for SNP50, SNP100, MSNP, and HMSNP were determined to be 25 µg/ml, 50 µg/ml, 200 µg/ml, and 200 µg/ml, respectively (Fig.1i). Morphology of all particles was assessed by TEM (Fig 1ii). With nontoxic doses, ICP-MS data showed that maximum particle uptake was achieved at 6 hours (Fig 2). Live/dead assay showed no statistically significant difference in apoptotic cell population for SNP100 and SNP50, whereas mesoporous and hollow SNPs showed a significant increase (P < 0.05) in the early apoptotic population (Fig.3). The CD80 surface marker expression increased significantly when treated with MSNP and MHSNP. No significant changes in membrane integrity, cell cycle, and phagocytic activity were observed after treating macrophages with the SNPs.

Conclusion: This study shows that there are differences in nontoxic doses of SNPs as a function of their physicochemical properties. At nontoxic dosages, there is no significant physiologically relevant adverse effect on the saturation of macrophages. The dose-dependent effect of SNPs as a function of physicochemical properties that influence the downstream immune function of macrophages will have important implications for their safe and effective use in drug delivery applications. Future directions of this study include understanding the effect of saturation on cytokine release and changes in molecular signaling pathways in different in vitro models.

References: 1. Janjua, T. I., Cao, Y., Yu, C. & Popat, A. Clinical translation of silica nanoparticles. Nature Reviews Materials vol. 6 1072–1074 (2021).
2. Wu, S. H., Hung, Y. & Mou, C. Y. Mesoporous silica nanoparticles as nanocarriers. Chemical Communications 47, 9972–9985 (2011).
3. Rieter, W. J. et al. Hybrid silica nanoparticles for multimodal imaging. Angewandte Chemie - International Edition 46, 3680–3682 (2007).

Acknowledgements: We acknowledge the National Institute of Health (NIH) for funding the project under grant no.
5R01ES024681.


Fig.1 (i) Dose-dependent toxicity of SNPs in RAW 264.7 cells, (ii) TEM images showing the size and morphology of the SNPs, (a) Stöber 50 SNPs, (b) Stöber 100 SNPs, (c) Mesoporous SNPs, (d) Hollow Mesoporous SNPs.



Fig.2 ICP-MS data showing the maximum amount of Si internalized at different time points. From the graphs, the 6h timepoint showed the maximum internalized silicon inside the cells.


Fig.3: Live-dead assay showed no significant change in live cell population upon saturation.