(W1030-02-11) Immunotoxicity of Silica Nanoparticles as a Function of Physicochemical Properties

Purpose: Silica nanoparticles (SiNPs) are a promising platform for drug delivery applications, which require an understanding of their biocompatibility, including their interactions with the immune system and peripheral blood cells. However, detailed studies for silica nanoparticle immunological safety profiles as a function of their physicochemical properties are lacking. Environmental and occupational immunotoxicology studies report that silica nanoparticles can induce lung inflammation in various in vivo and in vitro models through mechanisms including oxidative stress, disruption of autophagy, and lysosomal dysfunction [1,2]. To fill this gap, we conducted a study investigating SiNPs’ interactions with various blood components using four types of SiNPs with variations in size, porosity, and density. These included spherical nonporous SiNPs with nominal average diameters of 50 and 100 nm (SNP50 and SNP100), mesoporous SiNPs with an average diameter of 100 nm (Meso100), and hollow mesoporous SiNPs with an average diameter of 100 nm (HMSNP100), respectively.

Methods: SNP50 and SNP100 were synthesized by the Stöber sol-gel method [3]. Meso100 and HMSNP100 were synthesized by the modified Stöber approach using the templating surfactant cetyltrimethylammonium bromide (CTAB) [4]. Particles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), nitrogen adsorption-desorption, and Fourier-Transform Infrared Spectroscopy (FTIR). Particle stocks were analyzed for endotoxin contamination by kinetic turbidity Limulus Amebocyte Lysate (LAL) assay. Hematological compatibility of these particles was assessed in fresh blood from healthy human donors by platelet aggregation, plasma coagulation, complement activation, and hemolysis assays. The effects of these particles on immune cell function were studied by leukocyte proliferation, human lymphocyte activation, and leukocyte procoagulant activity assays employing peripheral blood mononuclear cells, and natural killer (NK) cell cytotoxicity assay utilizing NK92 effector and HepG2 target cell lines. The concentrations of SiNPs tested in vitro were 10, 50, 100, and 500 mg/mL, comparable to that of other human intravenous dosing ranges of nanocarriers currently used for drug delivery in the clinic.

Results: Successful particle synthesis was determined by DLS and TEM analysis, confirming objective morphology and size. The surface area surface areas of SNP50, SNP100, Meso100, and HMSNP100 were 58.2 m²/g, 35.7 m²/g, 1,219.1 m²/g, and 896.0 m²/g, respectively. FTIR confirmed complete removal of CTAB from the mesoporous particles, ensuring no immunotoxicity was observed as a result of residual CTAB. Endotoxin levels in the nanoparticle stocks were undetectable by the LAL assay ( < 0.1 EU/mL). Hematological compatibility assessment resulted in toxicities dependent on physicochemical properties at the highest nanoparticle concentration tested. HMSNP100 at 500 mg/mL was observed to induce platelet aggregation and prolong plasma coagulation time. SNP50 and SNP100 induced low levels of complement activation at 500 mg/mL. Hemolysis was observed for SNP50, SNP100, and HMSNP100 at 500 mg/mL only. The effects of these particles on immune cell function were also dependent on SiNP physicochemical properties. No particles induced leukocyte proliferation alone, but all particle types resulted in dose-dependent inhibition of mitogen-induced leukocyte proliferation. SNP50 and SNP100 at 500 mg/mL were the only samples to inhibit lymphocyte proliferation in the human lymphocyte activation assay. This suggests a different mechanism of immunosuppression between mesoporous and nonporous SiNPs. Leukocyte procoagulant activity (PCA) was observed only with Meso100 at 500 mg/mL, inducing plasma coagulation after nanoparticle-treated peripheral blood mononuclear cells were added to fresh plasma from the same donors. This was confirmed by analyzing for PCA complex on the surface of monocytes, consisting of elevated levels of tissue factor (TF) and surface exposed phosphatidylserine (PS), via flowcytometry. Indeed, only Meso100-treated monocytes showed high levels of both TF and PS surface expression. Lastly, the cytotoxic activity of NK cells towards the target cancer line was only inhibited after exposure to SNP50 at 500 mg/mL. Conversely, the cytotoxic activity of NK cells increased after incubation with supernatant isolated from peripheral blood mononuclear cells cultured with SiNPs at 100 mg/mL, for all particles. This suggests that NK cells can be indirectly activated by SiNPs exposure in vivo through cytokine signaling by other immune cells.

Conclusion: Our findings suggest that at high concentrations (500 mg/mL), corresponding to the in vivo human dose of 40 mg/kg, SiNPs demonstrated an array of immunotoxic effects dependent on their physicochemical properties. However, all four types of SiNPs studied were not immunotoxic at lower in vitro concentrations (100 mg/mL), corresponding to clinically relevant doses comparable to that of nanocarriers in nanomedicines currently approved for use in the clinic. These findings are promising for using SiNPs for the systemic delivery of bioactive and imaging agents.

References: 1 Wang, M.; Li, J.; Dong, S.; Cai, X.; Simaiti, A.; Yang, X.; Zhu, X.; Luo, J.; Jiang, L. H.; Du, B.; Yu, P.; Yang, W. Silica nanoparticles induce lung inflammation in mice via ROS/PARP/TRPM2 signaling-mediated lysosome impairment and autophagy dysfunction. Part Fibre Toxicol 2020, 17(1):23. DOI: 10.1186/s12989-020-00353-3.
2 Zhou, X.; Jin, W.; Ma, J. Lung inflammation perturbation by engineered nanoparticles. Front Bioeng Biotechnol 2023, 11:1199230. DOI: 10.3389/fbioe.2023.1199230.
3 Stöber, W.; Fink, A.; Bohn, E. Controlled growth of monodisperse silica spheres in the Micron Size Range. J Colloid Interface Sci 1968, 26(1):62–69. DOI: 10.1016/0021-9797(68)90272-5.
4 Trzeciak, K.; Chotera-Ouda, A.; Bak-Sypien, I. I.; Potrzebowski, M. J. Mesoporous Silica Particles as Drug Delivery Systems-The State of the Art in Loading Methods and the Recent Progress in Analytical Techniques for Monitoring These Processes. Pharmaceutics 2021, 13(7):950. DOI: 10.3390/pharmaceutics13070950.

Acknowledgements: Support was provided by NIH Grant R01ES024681 and by the National Cancer Institute under the contract 75N91019D00024.