GlycoRNA-rich, neutrophil membrane-coated, siMT1-loaded nanoparticles mitigate abdominal aortic aneurysm progression by inhibiting the formation of neutrophil extracellular traps

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening vascular condition. Currently, there are no clinically available pharmacological interventions that can stop the progression of AAA, primarily due to the incomplete understanding of its pathogenesis and the absence of effective drug delivery systems. The present study aimed to develop a targeted therapy for AAA through a nanomedicine approach involving site-specific regulation of neutrophil extracellular trap (NET)-related pathological vascular remodeling. We found that metallothionein 1 (MT1) was upregulated in AAA lesions in both humans and mice. MT1 also facilitated the formation of NETs and subsequently induced phenotypic transformation and apoptosis in vascular smooth muscle cells. Additional in vivo studies revealed that the glycoRNA-rich membranes coated siMT1-loaded poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) nanoparticles (GlycoRNA-NP-siMT1) effectively delivered siMT1 to AAA lesions, thereby inhibiting abdominal aortic dilation. Mechanistically, GlycoRNA-NP-siMT1 mitigated pathological remodeling of the abdominal aorta by reducing neutrophil infiltration and inhibiting the formation of NETs. Our study indicates that MT1 facilitates the progression of AAA by modulating the formation of NETs. Furthermore, GlycoRNA-NP-siMT1 show an inhibitory effect on AAA progression through a dual mechanism: they competitively inhibit neutrophil infiltration and release siMT1, which subsequently suppresses NET formation.

Keywords: Abdominal aortic aneurysm; GlycoRNA; Metallothionein 1; Nanoparticles; Neutrophil extracellular traps.

Graphical abstract

Fig. 1

MT1 expression in human AAA tissues. (A) Three-dimensional reconstructed CT image of AAA. (B) Human AAAs were subjected to immunofluorescence (IF) staining for detecting MT1 (red) and α-SMA (green, VSMC marker) expression, and the nuclei were counterstained with DAPI. Scale bar = 100 μm. (C) Quantification of MT1 mean fluorescence intensity (MFI) (n = 8). (D–J) The mRNA expression levels of TNF-α, MMP2, MMP9, ACTA2, TAGLN, BCL-2, and BAX were assessed by RT-qPCR (n = 4). Data are expressed as mean ± SD. P-values were calculated by unpaired Student's t-test. ∗∗P < 0.01 and ∗∗∗P < 0.001. MT1 = Metallothionein 1; AAA = abdominal aortic aneurysm; α-SMA = α-smooth muscle actin; VSMCs = vascular smooth muscle cells; DAPI = 4′,6-Diamidino-2′-phenylindole; MFI = mean fluorescent intensity; TNF-α = tumor necrosis factor-α; MMP2/9 = matrix metallopeptidase 2/9; ACTA2 = Actin Alpha 2; TAGLN = transgelin; BCL-2 = B-cell lymphoma-2; BAX = BCL-2 associated X protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

MT1 silencing inhibits the formation of NETs. (A) Treated neutrophils were subjected to IF staining for CitH3 (red) and MPO (green, both are NET markers) expression at 8 h, and the nuclei were counterstained with DAPI. Scale bar = 50 μm. (B and C) Quantification of Quantification of the fluorescence intensity (n = 4). (D) Treated neutrophils were stained with cytoROS probes (green, scale bar = 50 μm). (E) Quantification of cytoROS MFI (n = 4). (F–I) Determination of the levels of inflammatory factors, including TNF-α, IL-1β, IL-6, and MCP-1, by ELISA. Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗∗P < 0.01 and ∗∗∗P < 0.001. NETs = neutrophil extracellular traps; CitH3 = citrullinated histone H3; MPO = myeloperoxidase; cytoROS = cytoplasmic reactive oxygen species; ELISA = enzyme linked immunosorbent assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Synthesis and characterization of GlycoRNA-NP-siMT1. (A and B) siMT1 degradation was assessed by gel electrophoresis. (c) Morphology of NPs was detected by TEM. (D and E) Particle size and zeta potential were assessed by DLS. (F and G) Particle structure stability test in PBS and serum. (H and I) Internalization of NPs was performed in ECs and VSMCs. Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. NPs; &&& P < 0.001 vs. HL60-NPs. HL60-NPs = HL60-NP-siMT1; G-NP = GlycoRNA-NP-siMT1; TEM = transmission electron microscopy; DLS = dynamic light scattering.

Fig. 4

In vivo targeting analysis of NPs. (A) Timeline of in vivo imaging. (B) Fluorescence degradation curve of NPs. (C) Detection of in vivo location of Cy5-labeled NPs (red) by IVIS. (D) Quantification of the fluorescence intensity of Cy5 (n = 6). (E) Detection of the location of Cy5-labeled NPs in the abdominal aorta (VSMC marker; green) by IF staining. Scale bar = 50 μm. (F) Quantification of the fluorescence intensity of Cy5 (n = 6). Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. PBS. &&& P < 0.001 vs. HL60-NPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Treatment with GlycoRNA-NP-siMT1 prevents AAA progression. (A) Survival rate curve (n = 20). (B) Quantification of the aneurysm incidence in each group (n = 20). (C) Quantification of the aneurysm rupture in each group (n = 20). (D) Representative image of the sacrificed aortas. (E and F) Quantification of the maximum aortic diameter and inner aortic diameter. (G) Elastin degradation detected by EVG staining. (H) Quantification of the elastin degradation score. Data are presented as mean ± SD. P values were determined using Kaplan-Meier analysis or one-way ANOVA with the post-hoc Bonferroni test. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. PBS. && P < 0.01, &&& P < 0.001 vs. G-NP-scramble. EVG = Elastic Van Gieson.

Fig. 6

GlycoRNA-NP-siMT1 treatment alleviates neutrophil infiltration in AAAs. (A) Timeline of chemical treatments. (B) Count of migrating cells (n = 4) (C) AAA sections were subjected to IF staining for detecting CD16 (red, neutrophils) and α-SMA (green, VSMC marker) expression, and the nuclei were counterstained with DAPI. Scale bar = 100 μm. (D) Quantification of CD16 MFI (n = 6). Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗∗∗P < 0.001 vs. PBS. & P < 0.05, &&& P < 0.001 vs. G-NP-scramble. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Treatment with GlycoRNA-NP-siMT1 inhibits the formation of NETs in AAAs. (A) AAA sections were subjected to IF staining for detecting CitH3 (red, NET marker) expression, and the nuclei were counterstained with DAPI. Scale bar = 100 μm. (B) Quantification of CitH3 MFI (n = 6). (C) AAA sections were subjected to IF staining for detecting DHE (red, ROS marker) expression, and the nuclei were counterstained with DAPI. Scale bar = 100 μm. (D) Quantification of DHE MFI (n = 6). (E–H) Determination of the level of inflammatory factors, including TNF-α, IL-1β, IL-6, and MCP-1, by ELISA. Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗∗∗P < 0.001 vs. PBS. && P < 0.01, &&& P < 0.001 vs. G-NP-scramble. DHE = Dihydroethidium. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Treatment with GlycoRNA-NP-siMT1 attenuates the phenotypic switching and apoptosis of VSMCs in AAA. (A) AAA sections were subjected to IF staining for detecting α-SMA (green, marker for phenotypic switching of VSMCs) expression, and the nuclei were counterstained with DAPI. Scale bar = 100 μm. (B) Quantification of α-SMA MFI (n = 8). (C) AAA sections were subjected to IF staining for TUNEL (red, apoptosis marker) and α-SMA (green, VSMC marker) expression, and the nuclei were counterstained with DAPI. Scale bar = 100 μm. (D) Quantification of TUNEL + cells (n = 8). Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. PBS. &&& P < 0.001 vs. G-NP-scramble. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

MT1 promotes the phenotypic switching and apoptosis of VSMCs by regulating the formation of NETs. (A) Timeline of chemical treatments. (B) Cells were subjected to IF staining for α-SMA (green, phenotypic switching marker for VSMCs) expression, and the nuclei were counterstained with DAPI. Scale bar = 50 μm. (C) Quantification of α-SMA MFI (n = 6). (D) Cells were subjected to IF staining for TUNEL (green, apoptosis marker) expression, and the nuclei were counterstained with DAPI. Scale bar = 50 μm. (E) Quantification of TUNEL⁺ cells (n = 6). Data are presented as mean ± SD. P values were determined using one-way ANOVA with the post-hoc Bonferroni test. ∗P < 0.05, ∗∗∗P < 0.001 vs. PBS. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

Safety assessment of GlycoRNA-NP-siMT1 treatment. (A) Timeline of safety assessment. (B) Results of blood cell analysis and blood biochemistry tests (n = 4). (C) H&E staining of the major organs in mice (n = 4). RBC = red blood cell; WBC = white blood cell; AST = aspartate transaminase; ALT = alanine Aminotransferase; BUN = Blood urea nitrogen; CR = creatinine. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)