Greater Plasma Protein Adsorption on Mesoporous Silica Nanoparticles Aggravates Atopic Dermatitis

Abstract

Purpose: The protein corona surrounding nanoparticles has attracted considerable attention as it induces subsequent inflammatory responses. Although mesoporous silica nanoparticles (MSN) are commonly used in medicines, cosmetics, and packaging, the inflammatory effects of the MSN protein corona on the cutaneous system have not been investigated till date.

Methods: We examined the greater plasma protein adsorption on MSN leads to serious inflammatory reactions in Dermatophagoides farinae extract (DFE)-induced mouse atopic dermatitis (AD)-like skin inflammation because of increased uptake by keratinocytes.

Results: We compare the AD lesions induced by MSN and colloidal (non-porous) silica nanoparticles (CSN), which exhibit different pore architectures but similar dimensions and surface chemistry. MSN-corona treatment of severe skin inflammation in a DFE-induced in vivo AD model greatly increases mouse ear epidermal thickness and infiltration of immune cells compared with the CSN-corona treatment. Moreover, MSN-corona significantly increase AD-specific immunoglobulins, serum histamine, and Th1/Th2/Th17 cytokines in the ear and lymph nodes. MSN-corona induce more severe cutaneous inflammation than CSN by significantly decreasing claudin-1 expression.

Conclusion: This study demonstrates the novel impact of the MSN protein corona in inducing inflammatory responses through claudin-1 downregulation and suggests useful clinical guidelines for MSN application in cosmetics and drug delivery systems.

Keywords: atopic dermatitis; claudin-1; colloidal silica; immunotoxicity; mesoporous silica; protein Corona.

Graphical abstract

Figure 1

Physicochemical properties of CSN and MSN (A) TEM images and EDS mapping of MSN and CSN. The scale bar is 50 nm. (B) XPS analysis of MSN and CSN showing identical surface atomic elements. (C) Size distributions of MSN and CSN showing identical sizes on the nanoscale. The mean size and electrical potential of MSN and CSN are identical. (D) MSN show greater surface areas due to increased porosity than CSN. (E) Capacity of FBS protein adsorption according to CSN and MSN concentration. (F) Electrophoretic bands of CSN and MSN reacted with proteins in FBS.

Figure 2

Physicochemical properties of Silica-Corona and BSA adsorption on Silica Corona. (A) Size distributions of CSN-FBS and BSA-blocked CSN show identical sizes in the nanoscale. (B) The electric potential of CSN-FBS and BSA-blocked CSN are identical. (C) TEM images show CSN Corona and additional adsorption of BSA on CSN Corona. The scale bar is 50 nm. (D) Size distribution of MSN-FBS and BSA-blocked MSN showing identical sizes in the nanoscale. (E) The electric potential of MSN-FBS and BSA-blocked MSN are identical. (F) TEM images show MSN Corona and additional adsorption of BSA on MSN-Corona. The scale bar is 50 nm. (G) Electrophoretic bands of CSN-Corona and MSN-Corona deactivated with BSA coating.

Figure 3

Cellular uptake of CSN and MSN in keratinocytes. (A) HaCaT cells treated with Alexa-CSN-Corona and Alexa-MSN-Corona showing different cellular uptake intensities after 6 h. (B) HaCaT cells pretreated with inhibitors of micropinocytosis (EIPA), clathrin-mediated endocytosis (CPZ) and caveolae-mediated endocytosis (GEN), showing that MSN-Corona uptake is mediated by clathrin and caveolin endocytosis after 6 h. (C) Florescence intensity (uptake) analysis of TNF-α- and IFN-γ-stimulated HaCaT treated with CSN-Corona or MSN-Corona. (D) Florescence intensity (uptake) analysis of TNF-α- and IFN-γ-stimulated HaCaT treated with MSN and inhibitor. (E) Schematic illustration of the difference between CSN-Corona and MSN-Corona uptake in HaCaT cells. The data are presented as the mean ± SD. **P < 0.01 and ***P < 0.001.

Figure 4

Cellular uptake of BSA-adsorbed MSN in keratinocytes. (A) HaCaT cells treated with MSN-Corona and BSA-blocked MSN-Corona, showing different cellular uptake intensities after 6 h. (B) Florescence intensity (uptake) analysis of TNF-α- and IFN-γ-stimulated HaCaT treated with MSN-Corona and BSA-adsorbed MSN. (C) Schematic illustration of the difference in intracellular uptake of the MSN-protein Corona and MSN-protein Corona blocked by BSA adsorption in HaCaT cells. The data are presented as the mean ± SD. ***P < 0.001.

Figure 5

MSN-Corona can induce the disruption of claudin-1. (A) HaCaT cells were treated with CSN-Corona, MSN-Corona, inactivated protein Corona-CSN, or inactivated protein Corona-MSN for 12 h. Cells were fixed and stained with DAPI prior to visualization of subcellular localization of claudin-1 (red) or nuclei (blue) using immunofluorescence analysis on a laser confocal microscope. Confocal image shows that damage to claudin-1 does not occur in the group treated with BSA-blocked MSN Corona. (B) Quantification of immunofluorescence for claudin-1. TNF-α and IFN-γ were used as positive controls. (C) To examine claudin-1 expression in CSN- or MSN-treated AD mice, the ears were excised on day 21. Whole cell lysates were analyzed using Western blotting (top). The claudin-1 expression levels were normalized to that of β-actin and quantified using the Image J software (bottom). The data are presented as the mean ± SD of three determinations. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Topical application of CSN-Corona and MSN-Corona does not affect healthy mouse skin. (A) The experimental scheme for topical application of CSN-Corona, MSN-Corona, or DFE. CSN-Corona, MSN-Corona, or DFE was applied six times for three weeks (n = 5). (B) Representative photomicrographs of ear sections stained with hematoxylin and eosin (H&E, scale bar = 40 µm). (C) Ear thickness was measured 24 h after CSN-Corona, MSN-Corona, or DFE application with a dial thickness gauge. Serum total IgE (D) and DFE-specific IgE (E) levels were measured using ELISA. (F) Schematic illustration of normal skin tissues upon MSN and CSN treatment. The data are presented as the mean ± SD. ***P < 0.001 compared with the control.

Figure 7

MSN-Corona exacerbate clinical features of AD skin inflammation compared to CSN. (A) DFE was applied to both ears twice a week for 4 weeks. CSN-Corona and MSN-Corona were applied six times for 3 weeks (n = 5). (B) Ear thickness was measured 24 h after DFE application with a dial thickness gauge. (C) Representative photomicrographs of ear sections stained with H&E or toluidine blue (scale bar = 40 µm). (D) Epidermal thickness (left), dermal thickness (middle), and number of eosinophils (right). (E) Number of mast cells (left) and serum histamine levels (right). (F) Schematic illustration of AD skin tissues upon MSN and CSN treatment. The data are presented as the mean ± SD of five determinations. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

Higher T cell immune responses to MSN in AD skin inflammation. Serum total IgE, DFE-specific IgE (A), and serum total IgG2a (B) levels were measured using ELISA. Blood samples of various groups, the vehicle (Ctrl), DFE plus the vehicle, and DFE plus nanoparticles, were collected by an orbital puncture on day 21. (C) CD4⁺ T cells in LNs of mice treated with CSN-Corona or MSN-Corona on day 21 after AD induction were analyzed by intracellular cytokine staining for FACS analysis. Numbers in quadrants indicate the percentages of cells expressing CD4⁺ IFN-γ, IL-4, or IL-17A. Statistical analysis of IFN-γ-, IL-4-, or IL-17-expressing CD4⁺ T cells in the LNs. (D) Expression of cytokines in the ears of CSN-Corona- or MSN-Corona-treated AD mice. To determine the cytokine levels in mice, the ears were excised on day 21. Gene expression was analyzed using qPCR. The gene expression levels were normalized to the β-actin level, and the values of fold-changes are presented. (E) Schematic illustration of T cell-mediated immune reactions from AD skin tissues upon MSN and CSN treatment. The data are presented as the mean ± SD of five determinations. *P < 0.05.