Colchicine delivered by a novel nanoparticle platform alleviates atherosclerosis by targeted inhibition of NF-κB/NLRP3 pathways in inflammatory endothelial cells

Figures

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  Fig. 1. Schematic illustration of the preparation of VHPK-PLGA@COL and the therapeutic paradigm and potential mechanisms. a After encapsulating COL in the nanoparticles, the VHPK peptide was subsequently added to target AS, forming VHPK-PLGA@COL. b These nanoparticles could accumulate in inflamed endothelial cells that overexpressed VCAM-1 and restrict the progression of AS by inhibiting NF-κB/NLRP3 pathways and reducing the secretion of proinflammatory cytokines such as IL-1β and IL-18. c In our study, six-week-old male ApoE − / − mice were divided into 5 groups (G1-G5). In G1, the normal group, mice were fed a normal chow diet (NCD). In G2 to G5, mice were fed a high-cholesterol diet (HCD) for 10 weeks followed by treatment with saline, VHPK-PLGA, COL, or VHPK-PLGA@COL, respectively, for 8 weeks

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  Fig. 2. Preparation of VHPK-PLGA@COL and confirmation of VHPK peptide binding. a Schematic illustration of the preparation of VHPK-PLGA@COL. b Investigation of VHPK peptide (labeled with FITC in green) on the surface of PLGA@COL (labeled with DiI in red) by CLSM (scale bar = 2 μm). c Quantitative analyses of the conjugation rate of VHPK peptide on PLGA@COL and VHPK-PLGA@COL by the FCM method. d Quantitative analysis of flow cytometry. e The VHPK peptide binding capacity (BC) and binding efficiency (BE) on the surface of VHPK-PLGA@COL

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  Fig. 3. Characterization of VHPK-PLGA@COL and drug release. a Appearance characteristics of COL, VHPK-PLGA and VHPK-PLGA@COL. b, c SEM and TEM images of VHPK-PLGA@COL. d, e The size distribution and zeta potentials of VHPK-PLGA and VHPK-PLGA@COL. f Average size and PDI of VHPK-PLGA@COL over 7 days. g UV‒vis–NIR absorbance spectra of VHPK-PLGA, COL and VHPK-PLGA@COL. h The absorbance of COL at different concentrations. i The corresponding concentration-absorbance intensity relationship at a wavelength of 353 nm. (j) The encapsulation efficiency (EE) and drug loading capacity (LC) of VHPK-PLGA@COL. k In vitro cumulative release of COL from VHPK-PLGA@COL in PBS solution at 4 °C and 37 °C, 100 rpm, and COL in PBS solution at 37 °C, 100 rpm. l Plasma concentration–time profiles of colchicine in rats after intravenous administration of colchicine and VHPK-PLGA@COL at an equivalent dose of 0.1 mg/kg colchicine. (mean ± SD, n = 3)

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  Fig. 4. In vitro cytotoxicity and blood compatibility analyses of VHPK-PLGA@COL. a, b CCK-8 assay on cell viability of HUVECs after treatment with COL and VHPK-PLGA@COL at various concentrations for 24 h and 48 h, respectively. c Live/dead cell-staining images of HUVECs after treatment for 24 h with COL and VHPK-PLGA@COL (colchicine: 0.8 µg/mL) as observed by CLSM. d, e FCM analysis of the rate of living HUVECs after treatment with COL and VHPK-PLGA@COL (colchicine: 0.8 µg/mL) for 24 h. f Images of the hemolysis test of VHPK-PLGA@COL at different concentrations after centrifugation. g Measurement of the absorbance of the supernatant at 540 nm in the hemolysis test. (n = 3, *P < 0.05, **P < 0.01***P < 0.001, ****P < 0.0001; ns = no significance)

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  Fig. 5. Targeting ability of PLGA@COL and VHPK-PLGA@COL in vitro. HUVECs with or without activation by TNF-α, TNF-α ( +) or TNF-α (-), were incubated with DiI-labeled PLGA@COL or VHPK-PLGA@COL for 2 h and images were observed with CLSM. For blocking analysis, activated cells were first treated with VHPK peptide solution followed by incubation with VHPK-PLGA@COL (middle column). (blue = nucleus, red = nanoparticles, scale bar = 50 μm)

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  Fig. 6. Targeting ability of VHPK-PLGA@COL in vitro: Time-point study. a CLSM images and b FCM results of activated HUVECs following incubation with DiI-labeled PLGA@COL or VHPK-PLGA@COL for different periods. c Quantitative results of data from flow cytometric analyses. (blue = nucleus, red = nanoparticles, scale bar = 50 μm, n = 3, **P < 0.01***P < 0.001)

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  Fig. 7. Targeting atherosclerotic plaques by VHPK-PLGA@COL in vivo. a Representative ex vivo fluorescence images of aortas and major organs, b Quantitative data of DiR fluorescent signals accumulated in the aortas, c Heatmap of DiR fluorescent signals in major organs at 24 h post intravenous injection (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). d CLSM images of aortic sinus sections prepared from atherosclerotic mice after intravenous injection of saline, DiR-labeled VHPK-PLGA or VHPK-PLGA@COL for 24 h. (blue = DAPI, green = VCAM-1, red = nanoparticles, P = atherosclerotic plaque, L = vessel lumen, scale bar = 50 μm)

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  Fig. 8. Biosafety evaluation of VHPK-PLGA@COL in vivo. a H&E staining of the heart, liver, spleen, lung, and kidney of ApoE − / − mice after different treatments. b Body weight and c hematological parameters and biochemical parameters of ApoE − / − mice after different treatments. (n = 5, Scale bar = 100 µm)

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  Fig. 9. Quantitative analysis of atherosclerotic plaques. a Digital images of the aortic arch (top) and en face micrographs of ORO-stained aortas (bottom) after 8 weeks of treatment. b Plaque area ratio (%) in the aortic arch; (c) Quantitation of the percentage of ORO-positive area compared with total luminal surface area (n = 5, *P < 0.05, **P < 0.01***P < 0.001, ****P < 0.0001; ns no significance)

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  Fig. 10. Assessment of the effect of VHPK-PLGA@COL on the stability of atherosclerotic plaques and measurement of plasma inflammatory cytokines in mice. a H&E and ORO-staining, and immunofluorescence studies on the expression of CD68 and MMP-9 on cross-sections of aortic sinus (scale bar = 200 μm). b-e Quantitative analysis of plaque area, lipid deposition area, and CD68 and MMP-9 mean fluorescence intensity in cross-sections of the aortic sinus. f The levels of proinflammatory factors (TNF-α, IL-1β, IL-18 and CRP) in the plasma of mice from different treatment groups were measured using ELISAs. (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns no significance)

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  Fig. 11. Inhibitory effect of VHPK-PLGA@COL on NF-κB/NLRP3 inflammasome pathways. a Western blot (WB) analysis on the expression of p65, NLRP3, caspase-1, IL-1β, and IL-18 proteins in the aortas of different groups of mice after 8 weeks of treatment. b-f The semi-quantification of WB data on p65, NLRP3, caspase-1, IL-1β, and IL-18 protein expressions in aorta (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no significance)