Materials

PLGA-PEG-COOH (PLGA (lactide:glycolide = 50:50, MW: 10000 Da); PEG (MW: 2000 Da)) was purchased from Xi’an Ruixi Biological Technology Co. (Xi’an, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HACAKA (Shanghai, China). Colchicine, polyvinyl alcohol (PVA; MW: 30,000–70,000 Da), the fluorescent dyes 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), 4′,6-diamidino-2-phenylindole (DAPI), N-(3-dimethylaminopropyl)-N’-ethylcarbodii-mide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were provided by Sigma‒Aldrich Corporation (St Louis, MO, USA). VHPK (VHPKQHRGGSKGC) and FITC-VHPK peptides were purchased from Qiangyao Biological Technology Co. (Suzhou, China). The Cell Counting Kit-8 (CCK-8) assay kit was purchased from Dojindo Laboratories (Mashiki-machi, Tabaru, Japan). Recombinant human tumor necrosis factor α (TNF-α) was purchased from NoVo Protein Scientific, Inc. (Canada). The anti-mouse CD68 antibody, anti-mouse VCAM-1 antibody, anti-mouse MMP-9 antibody, anti-mouse NLRP3 antibody, anti-mouse IL-1β antibody, anti-mouse IL-18 antibody, anti-mouse p65 antibody, and anti-mouse caspase-1 antibody were purchased from Cell Signaling Technology (MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). Oil red O (ORO) and hematoxylin and eosin (H&E) were purchased from Servicebio (Wuhan, China). Human umbilical vein endothelial cells (HUVECs) were purchased from Science Cell (Carlsbad, CA).

Preparation of VHPK-PLGA@COL and confirmation of VHPK peptide bindingPreparation of VHPK-PLGA@COL

PLGA@COL was prepared using a modified double-emulsion method (W/O/W method) [28, 29]. According to this method, 50 mg of PLGA-PEG-COOH and 25 mg of colchicine were fully dissolved in 2 mL of chloroform (CHCl3) as the oil phase. Then, 200 μL of double-distilled water was added to serve as the inner aqueous phase, and the mixture was emulsified with a sonicator (Sonics & Materials Inc., Newtown, Connecticut, USA) to obtain a primary emulsion. Subsequently, 5 mL of 4% PVA solution was added for the second sonication cycle to form a W/O/W double emulsion. Ten milliliters of 2% isopropanol solution was then added, and the solution was magnetically stirred for 4 h until the organic solvents had evaporated completely and the surfaces of the NPs solidified. Finally, PLGA@COL was purified by centrifugation (10,000 rpm, 7 min).

VHPK-PLGA@COL was fabricated by the carbodiimide method [30]. Briefly, excess EDC and NHS at a molar ratio of 2:1 were added to 0.1 M MES buffer solution (pH = 5.2) to activate the carboxyl group of PLGA@COL, and the mixture was allowed to react on a shaker for 2 h. The unreacted EDC and NHS were removed by centrifugal washing. Then, the activated PLGA@COL and 5 mg of VHPK peptide were dispersed in 0.1 M MES buffer solution (pH = 8) and reacted in a shaker for 12 h. Finally, VHPK-PLGA@COL was rinsed with double-distilled water three times to remove the unreacted materials.

DiI- or DiR-labeled PLGA@COL or VHPK-PLGA@COL was added to an appropriate amount of DiI or DiR when dissolving PLGA-PEG-COOH. We fabricated FITC-labeled NPs using a similar method except we replaced the VHPK peptide with FITC-VHPK peptide. VHPK-PLGA was prepared using the same procedures described above without the addition of colchicine in the first step as a blank control.

Confirmation of VHPK peptide binding

To confirm the conjugation of the VHPK peptide to PLGA@COL, VHPK-PLGA@COL was visualized by observing the colocalization of DiI-labeled PLGA@COL (red) and FITC-labeled VHPK peptide (green) using confocal laser scanning microscopy (CLSM; A1R, Nikon, Tokyo, Japan). The carrier rate of the VHPK peptide was analyzed by flow cytometry (FCM; FACS Vantage SE, Becton Dickinson, San Jose, CA, USA). To further evaluate the density of VHPK peptide on VHPK-PLGA@COL, the concentration of VHPK peptide in the supernatant was determined using high-performance liquid chromatography (HPLC, Kromasil 100-5C18: 5 µm, 4.6 mm × 250 mm; Temperature: 25 ℃; Mobile phase: 0.1% Trifluoroacetic Acid in Acetonitrile and 0.1% Trifluoroacetic Acid in water; Flow Rate: 1.0 ml/min; Run Time: 20 min; Wavelength: 220 nm). The VHPK peptide binding capacity (BC) and binding efficiency (BE) were calculated as follows:

$$BC\,(\% )\, = \,\frac}\, \times \,100\%$$

$$BE\,(\% )\, = \,\frac}\, \times \,100\%$$

The mass of VHPK peptide on nanoparticles = mass of VHPK peptide used – mass of VHPK peptide in the supernatant.

Characterization of VHPK-PLGA@COL

The morphology and structure of VHPK-PLGA@COL were observed using scanning electron microscopy (SEM; Hitachi S-3400N, Hitachi, Ltd., Tokyo, Japan) and transmission electron microscopy (TEM; Hitachi H-7600, Hitachi, Ltd., Tokyo, Japan). The size, polydispersity indexes (PDIs), and zeta potentials of VHPK-PLGA and VHPK-PLGA@COL were measured by a dynamic light scattering detector (DLS, Malvern Instruments, Malvern, UK). To test the stability of VHPK-PLGA@COL, the size and PDI were monitored using DLS in phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) for 7 d.

The standard curve of colchicine dissolved in double-distilled water was established by measuring the absorbance of solutions with different concentrations with a UV‒vis–NIR spectrophotometer at a wavelength of 353 nm. The concentration of colchicine was calculated based on the corresponding absorbance of the UV spectrum at 353 nm. The drug loading capacity (LC) and encapsulation efficiency (EE) were calculated as follows:

$$LC\,(\% )\, = \,\frac}\, \times \,100\%$$

$$EE\,(\% )\, = \,\frac}\, \times \,100\%$$

The mass of COL encapsulated in nanoparticles = mass of COL used—mass of COL in the supernatant.

Drug release analysisIn vitro drug release analysis

The cumulative release of colchicine from VHPK-PLGA@COL was investigated by adding the nanoparticles into a dialysis bag (MWCO 3500 Da) and immersing it in 50 mL of PBS (pH 7.4) at 37 °C or 4 °C with shaking at 100 rpm. At specific time intervals (0, 0.5, 1, 1.5, 2, 2.5, 3, 6, 10, 16, 20, 24, 30, 39, and 48 h), 1 mL of sample was withdrawn from the buffer solution and replaced with an equal volume of fresh PBS. As a control, an equal amount of free colchicine was added to a dialysis bag (MWCO 3500 Da) at 37 °C with shaking at 100 rpm. The cumulative release of colchicine was calculated according to the standard curve.

In vivo drug release analysis

Male SD rats were purchased from the Animal Center of Chongqing Medical University. Rats were randomly divided into two groups (n = 3 for each group). Colchicine and VHPK-PLGA@COL were intravenously administered via the tail vein at an equivalent dose of 0.1 mg/kg colchicine. Blood samples (200 µL) were drawn from the carotid vein at the preset time points, e.g., 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, and 48 h, after intravenous administration, and centrifuged immediately at 3000 rpm for 5 min to obtain plasma. The plasma was placed in a 3 KDa centrifugal filter and centrifuged for 10 min at 10,000 rpm to separate free colchicine from plasma. The ultrafiltrate (50 µL) was placed in a glass tube with 50 µL of the internal standard (20 ng/ml tegafur), and 2 ml of n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) was added. The mixture was vortexed for 3 min and centrifuged at 3500 rpm for 10 min. Then, the upper organic layer was decanted into another tube and evaporated to dryness at 40 ℃ under a gentle stream of nitrogen. The residue was reconstituted in 50 µL of mobile phase and then was injected into a liquid chromatography–tandem mass spectrometer (LC–MS/MS) to measure the colchicine concentration. The LC–MS/MS system consisted of an HPLC (Agilent Technologies, Palo Alto, CA, USA) and a mass spectrometer (MS, Applied Biosystems Sciex, Ontario, Canada) using electrospray ionization (ESI). Chromatography was performed on a Zorbax Extend C18 column (5 µm, 150 mm × 4.6 mm i.d. Agilent Technologies) maintained at 40 ℃ with a mobile phase of formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) at a flow rate of 1.1 ml/min. For quantitative analysis, the MS was run in multiple reaction monitoring (MRM) mode. The MRM transitions for colchicine were 400.1 → 358.3 (m/z) and for tegafur were 200.5 → 130.9 (m/z).

Cell culture and establishment of the inflammatory cell model

HUVECs were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and incubated at 37 °C in a 5% CO2 atmosphere. Cultured cells in the logarithmic growth phase were used for cell experiments. Inflammatory endothelial cells were induced by incubation with 20 ng/mL TNF-α [4] at 37 °C for 24 h.

Establishment of the mouse model of atherosclerosis

Six-week-old homozygous male apolipoprotein E knockout C57BL/6 mice (ApoE − / − mice) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (License SCXK 2019e0008), quarantined, and acclimatized for one week before the experiments. All mice were subjected to a 12 h light/dark cycle under specific pathogen-free conditions at 27 °C and properly handled in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Chongqing Medical University. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University. ApoE − / − mice were fed a high-cholesterol diet (HCD; 40% fat, 40% carbohydrate, and 20% protein) with a high level of cholesterol (D12108C-high-fat rodent diet with 1.25% cholesterol, FBSH, Shanghai, China) for 10 weeks [4, 31] to induce atherosclerotic plaque formation in the mouse aortic region and establish an atherosclerotic mouse model. As the normal group, six-week-old ApoE − / − mice were fed a normal chow diet (NCD; 10% fat, 70% carbohydrate, and 20% protein).

In vitro cytotoxicity and blood compatibility tests of VHPK-PLGA@COLCytotoxicity test

The toxicity of colchicine and VHPK-PLGA@COL was detected by CCK-8 assays, live/dead cell staining, and FCM. First, HUVECs were seeded in a 96-well plate at a density of 1 × 104 cells per well and incubated for 24 h. The cells were treated with colchicine or VHPK-PLGA@COL at different concentrations (0.1, 0.2, 0.4, 0.8, 1, 30 and 50 μg/mL) for 24 h and 48 h, respectively. Cell viability was measured using a CCK-8 assay. In addition, HUVECs (5 × 105) were seeded in laser confocal cell culture dishes and cultured for 24 h, followed by treatment with 0.8 µg/mL colchicine and VHPK- PLGA@COL for 24 h. Then, the cells were stained with M5 HiPer Calcein AM/PI and observed by CLSM (Beijing, China). Meanwhile, the cell survival rate following drug treatment for 24 h was determined by FCM.

Blood compatibility test

For the hemolysis evaluation, fresh blood was collected from anesthetized C57BL/6 mice with anticoagulant containing ethylenediaminetetraacetic acid (EDTA), and the whole blood was diluted with saline to obtain diluted whole blood. Twenty microliters of the diluted whole blood was added to 1 mL of VHPK-PLGA@COL solution at various concentrations (0.4, 0.8, 2, 3, 4, and 5 µg/mL) and incubated at 37 °C for 1 h. Red blood cells in saline were used as negative controls, and those in double-distilled water were regarded as positive controls. Subsequently, the supernatant of each sample was collected by centrifugation (3000 rpm, 5 min), and the absorbance at 540 nm was measured using a microplate reader.

Analysis of the targeting ability of VHPK-PLGA@COLTargeting ability analysis in vitro

The ability of VHPK-PLGA@COL to target HUVECs was evaluated using both CLSM and FCM. The inactivated and activated cells (induced by 20 ng/mL TNF-α) were seeded into laser confocal cell culture dishes and cultured for 24 h. Then, the original medium was replaced with fresh medium containing 0.4 µg/mL DiI-labeled VHPK-PLGA@COL or PLGA@COL and incubated for another 2 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 15 min and stained with DAPI solution for another 10 min, followed by washing twice with PBS. Cell images were captured by CLSM. For the blocking experiment, activated HUVECs were cultured with medium containing free VHPK peptide solution overnight, followed by incubation with medium containing 0.4 µg/mL DiI-labeled VHPK-PLGA@COL for 2 h. Cell images were captured by CLSM. For dynamic uptake analysis, activated HUVECs were incubated with 0.4 µg/mL DiI-labeled VHPK-PLGA@COL or PLGA@COL for various periods (0, 0.5, 2, and 4 h), and the uptake of VHPK-PLGA@COL or PLGA@COL by HUVECs was quantitatively determined and analyzed by FCM.

In vivo targeting analysis

To determine the biodistribution and targeting capability of VHPK-PLGA@COL in vivo, equal volumes of DiR-labeled VHPK-PLGA@COL and PLGA@COL or saline as the control group were intravenously administered to ApoE − / − atherosclerotic mouse models (3 mice/group). After 24 h, the aortas from the aortic root to the bifurcation of the iliac artery, hearts, livers, spleens, lungs, and kidneys were dissected from different groups of mice and imaged by an in vivo imaging system (IVIS, Perkin Elmer, U.K.). In addition, sections of the isolated aortic sinuses were directly immunofluorescence staining of VCAM-1 (green) followed by staining with DAPI solution to determine the distribution of VHPK-PLGA@COL and PLGA@COL by CLSM.

Animal experimentsAnimal experimental protocol

As illustrated in the scheme (Fig. 1c), six-week-old male ApoE − / − mice were randomly and investigator-blindly divided into 5 groups (G1-G5: normal, control, VHPK-PLGA, colchicine, VHPK-PLGA@COL; 5 mice/group). Mice in G1, the normal group, were fed a normal chow diet (NCD) throughout the experimental period. All mice from G2 to G5 were first fed a high-cholesterol diet (HCD) for 10 weeks to establish the atherosclerotic model followed by treatment with saline as the control (G2), VHPK-PLGA (G3), colchicine (G4), and VHPK-PLGA@COL (G5), respectively.

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

The indicated formulations were administered intravenously via the tail vein every two days for 8 weeks at a dosage of 0.1 mg/kg colchicine and an equivalent amount of saline. The colchicine dosage used was based on previously published studies [32, 33]. Upon termination of the study, the body weights of mice were recorded, and the animals were sacrificed under anesthesia. Blood was collected in EDTA spray-coated tubes and centrifuged at 3000 rpm for 5 min to collect plasma. The aortas from the heart to the iliac bifurcation and organs were carefully dissected and fixed with 4% paraformaldehyde solution.

In vivo biosafety evaluation of VHPK-PLGA@COL

Hematological parameters, including red blood cells (RBCs), platelets (PLTs), white blood cells (WBCs), and hemoglobin (HGB) content, in blood samples collected from different groups of mice were analyzed, respectively. Meanwhile, biochemical parameters in the corresponding plasma samples, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), blood urea nitrogen (BUN), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were also evaluated accordingly. In addition, the major organs of mice, including heart, liver, spleen, lung, and kidney, were harvested and fixed with 4% paraformaldehyde for H&E staining.

Evaluation of the in vivo anti-atherosclerotic effect of VHPK-PLGA@COLQuantitative analysis of the atherosclerotic plaques

Digital images of the aortic arch were obtained after the aortas were dissected from the mice. The plaque shape, size, and distribution throughout the entire aorta were observed intuitively with gross pathological specimens. The whole aorta was longitudinally opened and stained with ORO, and photos of the stained aortas were quantitatively analyzed by ImageJ to evaluate the therapeutic efficacy of the different formulations.

Assessment of the stability of atherosclerotic plaques

To assess stability of the atherosclerotic plaques, sections of the aortic sinus were stained with H&E and ORO and incubated with antibodies, including anti-CD68 and anti-matrix metalloproteinase-9 (MMP-9), respectively, for immunofluorescence analysis. Then, the sections were visualized using CLSM. Furthermore, quantitative analysis of atherosclerotic plaques or the positive area was determined by ImageJ.

Quantification of inflammatory cytokines in plasma

The levels of TNF-α, IL-1β, IL-18, and CRP in the plasma of mice were analyzed and quantified using commercial ELISA kits according to the manufacturer’s instructions.

Western blot

Total proteins were extracted from entire aortas dissected from different groups of mice. The protein concentration was determined using BCA assay. Equivalent amounts of denatured protein samples were separated by 12% SDS‒PAGE, transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA), and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies against NF-κB p65, NLRP3, caspase-1, IL-1β, IL-18, α-Tubulin, and GAPDH, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. The blot signals were visualized by enhanced chemiluminescent (ECL) reagents and detected through a Bio-Rad imaging system (Bio-Rad, USA). The expression level of the target proteins was semi-quantified by measuring the relative gray value of each target protein band with the corresponding GAPDH or α-Tubulin as internal control for the normalization of data.

Statistical analysis

All statistical analysis were performed using GraphPad Prism (version 8.02). One-way analysis of variance (ANOVA) was performed for multiple comparisons. All data are displayed as the mean ± SD and P values < 0.05 were considered statistically significant.