Cytotoxicity Assessment of Surface-Modified Magnesium Hydroxide Nanoparticles

Abstract

Magnesium-based nanoparticles have shown promise in regenerative therapies in orthopedics and the cardiovascular system. Here, we set out to assess the influence of differently functionalized Mg nanoparticles on the cellular players of wound healing, the first step in the process of tissue regeneration. First, we thoroughly addressed the physicochemical characteristics of magnesium hydroxide nanoparticles, which exhibited low colloidal stability and strong aggregation in cell culture media. To address this matter, magnesium hydroxide nanoparticles underwent surface functionalization by 3-aminopropyltriethoxysilane (APTES), resulting in excellent dispersible properties in ethanol and improved colloidal stability in physiological media. The latter was determined as a concentration- and time-dependent phenomenon. There were no significant effects on THP-1 macrophage viability up to 1.500 μg/mL APTES-coated magnesium hydroxide nanoparticles. Accordingly, increased media pH and Mg²⁺ concentration, the nanoparticles dissociation products, had no adverse effects on their viability and morphology. HDF, ASCs, and PK84 exhibited the highest, and HUVECs, HPMECs, and THP-1 cells the lowest resistance toward nanoparticle toxic effects. In conclusion, the indicated high magnesium hydroxide nanoparticles biocompatibility suggests them a potential drug delivery vehicle for treating diseases like fibrosis or cancer. If delivered in a targeted manner, cytotoxic nanoparticles could be considered a potential localized and specific prevention strategy for treating highly prevalent diseases like fibrosis or cancer. Looking toward the possible clinical applications, accurate interpretation of in vitro cellular responses is the keystone for the relevant prediction of subsequent in vivo biological effects.

Figure 1

Mg(OH)₂-NPs generated by precipitation are shaped as hexagonal platelets. (A) NTA-determined size distribution of Mg(OH)₂-NPs and (B) TEM images of Mg(OH)₂ hexagonal NPs.

Figure 2

Cytotoxicity effect of Mg(OH)₂-NPs suspensions (2.500–19.5 μg/mL) for 48 h in different cell lines HDF, HUVEC, HPMEC, PK84, THP-1, and ASCs. According to the ISO 10993-5 standard values <70% are considered toxic.

Figure 3

Mitochondrial activity of THP-1 cells exposed to Mg(OH)₂-NPs with and APTES functionalization. According to the ISO 10993-5 standard values <70% are considered toxic.

Figure 4

APTES-Mg(OH)₂-NPs in acellular conditions (culture medium) and incubated at 37 °C for 5 min, 15 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. The pH measured at all the time points showed a significant increase upon exposure to 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, and 19.5 μg/mL compared to the control media.

Figure 5

Dissolution potential of APTES- Mg(OH)₂-NPs is reached almost immediately. APTES-Mg(OH)₂-NPs in acellular conditions (culture medium), incubated at 37 °C for 5 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. The Mg²⁺ concentration showed a significant increase at all the time points compared to the control media (A) and only limited changes over time (B). (****p < 0.0001).

Figure 6

Concentration-dependent NPs dissolution potential reflects in concentration-dependent Mg²⁺ release. APTES-Mg(OH)₂-NPs (2500–19.5 μg/mL) in acellular conditions (culture medium), incubated at 37 °C for 48 h. The Mg²⁺ concentration showed a significant increase compared to the control media (A) and the range of nanoparticle dissolution potential (B) (****p < 0.0001).

Figure 7

Alkaline pH and Mg²⁺ concentration in supernatant corresponding to 2500–19.5 μg/mL NPs concentration range do not exhibit cytotoxic effects in THP-1 macrophages. APTES-Mg(OH)₂-NPs (2500–19.5 μg/mL) were resuspended in acellular conditions (culture medium) for 48 h at 37 °C. After centrifugation, THP-1 macrophages were exposed to respective supernatants for 48 h at 37 °C. Increased Mg²⁺ levels showed no toxic effects, while alkaline pH affected THP-1 mitochondrial viability (**p < 0.0099).

Figure 8

Mg²⁺ dosage in supernatant, which is 2500–19.5 μg/mL NPs concentration range does not reach the IC₅₀ concentration indicated by the MgCl₂ standard curve. THP-1 macrophages were exposed to MgCl₂ (1–0.001 M, 10-fold serially diluted) for 48 h at 37 °C. IC₅₀ concentration of 50 mmol/L was indicated in an MTT assay. The concentration of Mg²⁺ in NPs supernatants (6.50–0.30 mmol/L) did not reach the IC₅₀ value hence it exhibited low adverse effects equivalent to ∼84% cell viability.

Figure 9

Functionalization process of the Mg(OH)₂-NPs with TEOS/APTES.