Cell Membrane-Coated Nanotherapeutics for the Targeted Treatment of Acute and Chronic Colitis

Abstract

Integrin α4β1 and α4β7 are overexpressed in macrophages and leukocytes and play important roles in mediating cell homing and recruitment to inflammatory tissues. Herein, to enhance the targeting ability of nanotherapeutics for inflammatory bowel disease (IBD) treatment, cyclosporine A-loaded nanoparticles (CsA NPs) were coated with macrophage membranes (MM-CsA NPs) or leukocyte membranes (LM-CsA NPs). In vitro experiments demonstrated that the physicochemical properties of the nanotherapeutics (e.g., size, zeta potential, polymer dispersity index, and drug release profiles) did not obviously change after cell membrane coating. However, integrin α4β1 and α4β7 were expressed in MM-CsA NPs and LM-CsA NPs, respectively, which significantly inhibited normal macrophage phagocytosis and obviously increased uptake by proinflammatory macrophages and endothelial cells. In vivo experiments verified that cell membrane-coated nanotherapeutics have longer retention times in inflammatory intestinal tissues. Importantly, LM-CsA NPs significantly mitigated weight loss, alleviated colon shortening, decreased disease activity indices (DAIs), and promoted colon tissue repair in acute and chronic colitis model mice. Furthermore, LM-CsA NPs significantly decreased the expression of inflammatory factors such as TNF-α and IL-6 and increased the expression of gut barrier-related proteins such as E-cadherin, ZO-1, and occludin protein in colitis mice.

Fig. 1.

Fabrication of cell membrane-coated CsA NPs for IBD treatment.

Fig. 2.

(A and B) Size distributions (A) and TEM images (B) of CsA NPs (a), MM-CsA NPs (b), and LM-CsA NPs (c). (C) Expression of β1 and β7 in cell membrane-encapsulated NPs and the cell membrane. (D and E) CLSM images of the colocalization of Cy5-labeled NPs (red) with DiO-labeled MMs (D) and lymphocyte membranes (LM) (E) (green) in RAW 264.7 cells. The cell nucleus was stained with DAPI (blue). (F) Drug release of CsA from CsA NPs (a), MM-CsA NPs (b), and LM-CsA NPs (c) incubated with or without 1.0 mM H₂O₂ in PBS.

Fig. 3.

Biodistribution of NPs in the colons of DSS-induced colitis mice. (A) In vitro fluorescence images of mouse colons at different time points after administration of free Cy5 (a), Cy5/CsA NPs (b), Cy5/MM-CsA NPs (c), and Cy5/LM-CsA NPs (d). (B) Region of interest analysis of fluorescence intensity in the colons at different time points. *, significantly different at P < 0.05; **, significantly different at P < 0.01.

Fig. 4.

In vivo therapeutic efficacy of NPs in DSS-induced acute colitis mice. (A) DSS and NP administration regimens in the acute mouse colitis model. (B and C) Body weights (B) and DAIs (C) of acute colitis model mice. (D and E) Representative macroscopic image of a colon (D) and analysis of colon length (E) on day 10. (F) Spleen index on day 10. (G) Analysis of MEICSs of mice in different groups. (H) Analysis of the histological score of colonic tissues. (I) Representative endoscopic image. (J) Representative H&E-stained histological sections. Red arrows indicate mucosal edema, black arrows indicate inflammatory cell infiltration, and blue arrows indicate crypt damage. *, significantly different at P < 0.05; **, significantly different at P < 0.01; ***significantly different at P < 0.001; ****significantly different at P < 0.0001.

Fig. 5.

Detection of the levels of inflammatory factors, MPO, and colon barrier-associated proteins in the colons of mice with acute colitis. (A) TNF-α, IL-6, and GAPDH levels in colon tissue were measured by Western blotting. (B and C) Relative expression of TNF-α (B) and IL-6 (C). (D) MPO levels in colon tissue. (E) Immunofluorescence results for ZO-1 and E-cadherin in the colonic tissue of mice with acute colitis (n = 4). Red, ZO-1; green, E-cadherin; blue, DAPI (nucleus). (F) The protein expression of occludin in the colon tissues of mice with acute colitis was detected by immunohistochemistry (n = 4). (G to I) Statistical analysis of ZO-1 (G), E-cadherin (H), and occludin protein (I) expression levels. *, significantly different at P < 0.05; **, significantly different at P < 0.01; ***significantly different at P < 0.001; ****significantly different at P < 0.0001.

Fig. 6.

In vivo therapeutic efficacy of NPs in DSS-induced chronic colitis model mice. (A) DSS and NP administration regimens in the chronic mouse colitis model. (B and C) Body weights (B) and DAIs (C) of acute colitis model mice. (D and E) Analysis of colon length (D) and the spleen index (E) at the end of the experiment. (F) Representative macroscopic image of the colon. *, significantly different at P < 0.05; **, significantly different at P < 0.01; ****, statistically significantly different at P < 0.0001.

Fig. 7.

Endoscopic analysis, histological evaluation, and proinflammatory cytokine levels in chronic colitis model mouse tissue. (A) Representative endoscopic image on the last day of the experiment. (B) Representative H&E-stained histological sections. Red arrows indicate mucosal edema, black arrows indicate inflammatory cell infiltration, and blue arrows indicate crypt damage. (C) TNF-α, IL-6, and GAPDH levels in colon tissue were measured by Western blotting. (D) Analysis of MEICSs. (E) Analysis of the histological score of colonic tissues. (F and G) Relative expression of TNF-α (F) and IL-6 (G). (H) MPO levels in colon tissue. *, significantly different at P < 0.05; **, significantly different at P < 0.01; ***, significantly different at P < 0.001; ****, significantly different at P < 0.0001.

Fig. 8.

Detection of the levels of colon barrier-associated proteins in the colon tissues of mice with chronic colitis. (A) Immunofluorescence results for ZO-1 and E-cadherin in the colonic tissue of mice with chronic colitis (n = 4). Red, ZO-1; green, E-cadherin; blue, DAPI (nucleus). (B) The expression of occludin protein in the colon tissue of mice with chronic colitis was detected by immunohistochemistry (n = 4). (C to E) Statistical analysis of ZO-1 (C), E-cadherin (D), and occludin protein (E) expression levels. **, significantly different at P < 0.01; ***, significantly different at P < 0.001; ****, significantly different at P < 0.0001.