ICAM-1-Targeted Liposomes Loaded with Liver X Receptor Agonists Suppress PDGF-Induced Proliferation of Vascular Smooth Muscle Cells
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The proliferation of vascular smooth muscle cells (VSMCs) is one of the key events during the progress of atherosclerosis. The activated liver X receptor (LXR) signalling pathway is demonstrated to inhibit platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation. Notably, following PDGF-BB stimulation, the expression of intercellular adhesion molecule-1 (ICAM-1) by VSMCs increases significantly. In this study, anti-ICAM-1 antibody-conjugated liposomes were fabricated for targeted delivery of a water-insoluble LXR agonist (T0901317) to inhibit VSMC proliferation. The liposomes were prepared by filming-rehydration method with uniform size distribution and considerable drug entrapment efficiency. The targeting effect of the anti-ICAM-T0901317 liposomes was evaluated by confocal laser scanning microscope (CLSM) and flow cytometry. Anti-ICAM-T0901317 liposomes showed significantly higher inhibition effect of VSMC proliferation than free T0901317 by CCk8 proliferation assays and BrdU staining. Western blot assay further confirmed that anti-ICAM-T0901317 liposomes inhibited retinoblastoma (Rb) phosphorylation and MCM6 expression. In conclusion, this study identified anti-ICAM-T0901317 liposomes as a promising nanotherapeutic approach to overcome VSMC proliferation during atherosclerosis progression.
KeywordsVascular smooth muscle cells Liver X receptor ICAM-1 Liposome
Studies in pathophysiology have shown that the activation, migration, and proliferation of vascular smooth muscle cells (VSMCs) play crucial roles in atherosclerosis. Notably, atherosclerosis is the primary cause of cardiovascular diseases and mortality in the industrialised nations [1, 2]. In general, VSMCs are quiescent and non-proliferative ; however, proliferation would be activated following vascular injury, which contributes to the pathogenesis of early atherosclerosis [4, 5]. Atherosclerotic lesions exhibit multiple molecular responses, including hyperglycemia, hypertension, and modified low-density lipoprotein expression, which damage endothelial cells and bind leukocytes and monocytes, causing lipoproteins to infiltrate the vascular intima . Subsequently, macrophages engulf the oxidised low-density lipoproteins (LDL) and become foam cells . As the inflammatory process continues, growth factors and cytokines, including the platelet-derived growth factors (PDGFs), promote VSMC migration from the medium to the intima and transition from a quiescent contractile state to an active synthetic state. This transition is the basis for atherosclerosis and restenosis .
Multiple approaches have attempted to weaken VSMC activation and proliferation. It has been demonstrated that LXR (LXRα and LXRβ) activation in macrophages and VSMCs regulated cholesterol homeostasis and inhibited mitogen-induced VSMC proliferation [9, 10]. LXRs (LXRα and LXRβ) are members of the nuclear hormone receptor family of transcription factors that inhibit VSMC proliferation and G1 → S phase progression by (i) preventing phosphorylation of retinoblastoma (Rb) protein at Ser807/811 and (ii) inhibiting the expression of minichromosome maintenance protein 6 (MCM6). Phosphorylation of Rb promotes cell cycle progression by releasing transcription factors, the S phase transcription factor E2F, that could induce gene expression required for DNA synthesis. The MCM6 is the machinery downstream of Rb phosphorylation that regulates the DNA replicative. Therefore, LXR agonists show great potential to inhibit VSMC proliferation. However, there hydrophobicity is the major barrier to their clinical use [10, 11].
Knowledge of atherosclerosis pathogenesis has provided opportunities for creative prevention strategies and treatments, including the use of nanotherapeutic approaches . The application of nanotechnology in medicine has provided credible strategies, including multimodal imaging and nanomedicine with sustained releases [13, 14, 15]. Phosphatidylcholine (PC) nanoparticles exhibit targeting ability for MRI and imaging of atherosclerotic plaques in mice . Nanoparticle-based drug delivery system (NDDs) is a promising therapeutic vector to improve the therapy efficiency for atherosclerosis treatment. NDDs overcomes many of the shortcomings that conventional therapies suffer from, including low water solubility and the inability to deliver drugs across a range of biological barriers . As far as we know, few reports on the targeted therapeutics based on NDDs have been presented to inhibit VSMC activation and proliferation. It has been found that after PDGF stimulation, the expression of intracellular adhesion molecule-1 (ICAM-1) by VSMCs was greatly upregulated, which binds multiple types of inflammatory cells . So, ICAM-1 could be identified as a marker for targeted drug delivery.
PC from eggs, cholesterol (Chol), PEGylated (Mw¼2000) 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG-mal) were purchased from Xi’an ruixi Biological Technology Co. Ltd. (Xian, China). Coumarin-6 was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Sephadex G-50 was obtained from GE Healthcare Life Sciences (WI, USA). Recombinant human PDGF-BB was purchased from PEPROTECH (Rocky Hill, USA). The LXRα agonist, T0901317, was purchased from Selleckchem (Boston, USA). Cell cycle analysis kits were obtained from BD Bioscience (San Jose, CA, USA). BrdU Cell Proliferation Detection Kits were obtained from KeyGen BioTECH (Nanjing, China). Mouse aortic smooth muscle (MOVAS) cells were purchased from Honsun Biologicals (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Anti-Rb antibodies targeting the total and phosphorylated (Ser 807/811) forms, as well as minichromosomal maintenance protein 6 (MCM6), and β-actin were purchased from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). CCK-8 and western blot detection reagents were purchased from Amersham Life Science Beijing Solarbio Science and Technology Co. Ltd. (Beijing, China). All chemicals were analytical or of high-performance liquid chromatography (HPLC) grade and used without additional purification.
Preparation of Multifunctional Liposomes
Preparation of Liposomes Loaded with T0901317 and Coumarin-6
Physicochemical characteristics of synthetic NP formulations
155.8 ± 2.38
0.133 ± 0.05
−1.9 ± 0.84
189.2 ± 2.74
0.173 ± 0.03
−18.1 ± 0.96
Preparation of Anti-ICAM-1 and T0901317 Liposome Conjugate
Anti-ICAM-1 antibodies were conjugated with T0901317 liposomes following previously described methods . Briefly, T0901317 liposomes (10 mg) and anti-ICAM-1 (20 μg) were dissolved in PBS (pH = 7.4) and stirred overnight in a glass bottle. Conjugates were then purified using a Sephadex G-50 (1 × 20 cm) gel filtration column.
The morphology and size of the anti-ICAM-1-T0901317- liposomes were measured by transmission electron microscopy (TEM; JEM-2100). Briefly, the liposome suspension was dropped on a copper grid and negatively stained with 1% phosphotungstic acid until dry. Hydrodynamic sizes, polydispersity (PDI), and zeta potential of the anti-ICAM-1-T0901317- liposomes and T0901317- liposomes were measured in aqueous solutions using a Zetasizer Nano ZS dynamic light scattering (DLS) instrument (DLS; Malvern Zetasizer 2000, Malvern, UK). UV spectrophotometry was used to determine whether liposomes were successfully bound to the target ICAM-1 antibodies. The absorption spectra were obtained using a UV-visible absorption spectrometer (UV-1601; Shimadzu; Kyoto, Japan) at wavelengths of 200–700 nm .
Measurement of In Vitro Entrapment Efficiency, Loading Efficiency, and Drug Release
To measure drug release from targeted and nontargeted liposomes, liposomes were diluted in 9 mL of PBS containing 0.1% sodium dodecyl sulphate (SDS; pH 7.4) and incubated in a vibrating water bath at 37 °C and 130 rpm. Following incubations of 0–50 h, samples were centrifuged at 20,000g for 15 min and mixed with equal volumes of PBS containing 0.1% SDS. The drug content in the supernatant was then measured by HPLC, and the cumulative T0901317 release from control or anti-OPN liposomes was plotted by the release ratio versus time.
MOVAS cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultivated in culture bottles at 37 °C and 5% CO2. Cells were treated with PDGF-BB at the final concentration of 20 ng/mL. The culture medium was replaced daily, and cells were sub-cultured at 100% confluence.
Treated cells were pre-incubated with PDGF-BB at the final concentration of 20 ng/mL for 24 h. Following digestion and centrifugation, cells were fixed with 4% paraformaldehyde and washed three times with ice-cold PBS containing 0.2% Triton X-100. Cells were then incubated for 1 h with primary anti-ICAM-1 antibodies in PBS containing 0.1% BSA 1. Cells were then washed three times in PBS and incubated for 1 h at 37 °C with FITC-conjugated secondary antibodies. Nuclei were stained with DAPI and washed three times. A confocal microscope (LSM510; Carl Zeiss; Germany) was used to analyse the expression of the target protein .
Cellular Uptake and Targeting Efficiency of Liposomes
MOVAS cells were cultured at 104 cells per well in confocal dishes and incubated for 24 h with PDGF-BB at the final concentration of 20 ng/mL. The medium was then replaced with the same containing 40 μL of T0901317- liposomes and anti-ICAM-1-T0901317 liposomes (1 mg lipid/mL), respectively. After 4 h, the medium was removed and cells were fixed with 4% paraformaldehyde. Nuclei were stained with DAPI for an additional 10 min. After washing the cells three times with PBS, confocal microscopy was performed (LSM510; Carl Zeiss; Germany) using two channels for DAPI and coumarin-6 (466 nm excitation, 504 nm detection). To validate the cellular uptake and targeting efficiency of liposomes, flow cytometry (Gallios; Beckman Coulter; CA, USA) was performed on 104 cells using an excitation wavelength of 466 nm.
Cell Proliferation Assay
Cellular proliferation was assessed using CCK-8 and BrdU Cell Proliferation Detection Kits. Cells were seeded in 96-well plates and treated with T0901317, anti-ICAM-1-T0901317-NPs, T0901317-NPs, and control liposomes for 24 h. Following incubation, medium was removed and cells were washed three times in PBS. The appropriate CCK-8 was added to each well and incubated for 4 h at 37 °C. The optical densities (OD) were determined at 450 nm using a microplate spectrophotometer.
Cell proliferation was also assessed by the number of nuclei that were DAPI stained and marked with BrdU. BrdU was used at a final concentration of 20 μmol/mL. The secondary antibody for BrdU was labelled TRITC. Images were obtained using an FV1200 microscope (Olympus Corporation; Japan).
Cell Cycle Analysis
Cells were cultured in six-well plates and incubated with T0901317, anti-ICAM-1-T0901317-NPs, T0901317-NPs, and blank liposomes for 24 h. Following incubation, medium was discarded and cells were washed three times in PBS. Treated cells were incubated with PDGF-BB for 24 h at a final concentration of 20 ng/mL, after which they underwent sequential incubation in trypsin, trypsin inhibitor and RNase buffer, and PI (propidium iodide) stain. Cells were incubated for 10 min on ice in the dark and assessed by flow cytometry (Gallios, Beckman Coulter; CA, USA).
Western Blot Analysis
Western blot analyses were performed as previously described . Total protein was extracted using 100 μL of radioimmunoprecipitation assay (RIPA) buffer and 1 μL of phenylmethylsulfonyl fluoride (PMSF) and quantified using the BCA Protein Assay (Pierce Biotechnology; MA, USA). Samples were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) bovine serum albumin (BSA) at room temperature for 30 min and incubated with appropriate primary and secondary antibodies. Blots were developed using the Supersignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology; MA, USA) and analysed using ImageJ software (National Institutes of Health; USA).
Data were analysed using SPSS.17 software and expressed as the means ± SEM. Assessments of statistical significance were performed using ANOVA and t tests (paired and unpaired). A P value of <0.05 was considered statistically significant.
Results and Discussion
The Scheme of Study Design
The scheme representing the subject of the article shows that the anti-ICAM-1-T0901317 liposomes were prepared by filming-rehydration method. Some specific materials contain phospholipid, cholesterol, T0901317, DSPE-PEG-mal, coumarin-6, and anti-ICAM-1. And the anti-ICAM-1-T0901317 liposomes can inhibit PDGF-induced proliferation of vascular smooth muscle cells effectively (Fig. 1).
Characterisation of Liposomes
Entrapment Efficiency, Loading Efficiency, and Drug Release
Lipid to T0901317 weight ratios of 5:1 and 10:1 were used in the evaporation process (50 mg total). The encapsulation efficiency was lower for the anti-ICAM-1-T0901317 liposomes when the ratio was 5:1, and drug loading was evaluated for all subsequent experiments. No significant differences in entrapment efficiencies were observed with antibody conjugation.
Influence of PDGF-BB on Expression of ICAM-1 in VSMCs
Cellular Uptake and Targeting Efficiency in Liposomes
Cell Proliferation Assessments by CCK-8 Assay and BrdU Immunofluorescence
Anti-ICAM-1-T0901317 Liposomes Inhibited PDGF-BB-Induced MOVAS Cell Proliferation and G1 → S Phase Progression
The effect of anti-ICAM-1-T0901317 liposomes on cell cycle progression was determined by flow cytometry. To analyse the effect on cell proliferation, it was necessary to analyse the effect of anti-ICAM-1-T0901317 liposomes on the cell cycle. Cells incubated with PDGF for 24 h preferentially accumulated in S phase (54.4 ± 2.8% in G0/G1 phase and 33.8 ± 2.0% in S phase; Fig. 7a). Treatment with T0901317 liposomes suppressed progression into S phase, causing an increase in the G0/G1 population (75.3 ± 3.2%). Additionally, cells incubated with anti-ICAM-1-T0901317 liposomes for 24 h primarily accumulated in G0/G1 phase (83.0 ± 1.3%). However, cells treated with control nanomaterials following incubation with PDGF-BB for 24 h did not exhibit altered cell cycles.
Anti-ICAM-1-T0901317 Liposomes Inhibit Phosphorylation of Rb and Expression of MCM6
VSMC proliferation plays an important role in the development of atherosclerosis, for which PDGF is a key mediator . The LXR agonist, T0901317, regulated lipid accumulation in VSMCs and inhibited smooth muscle cell proliferation by preventing the phosphorylation of Rb. Thus, T0901317 may play a protective role in preventing atherosclerotic plaque formation [17, 28]. ICAM-1, which is highly expressed following PDGF-BB treatment, was chosen as the target of sustained-release liposomes containing T0901317. The goal was to inhibit smooth muscle cell proliferation [29, 30]. Liposomes were used since they have many advantages, including targeted diagnoses and therapies, sustained drug release, and suitable hydrophobicities. The physicochemical characteristics of liposomes were determined by assessing their sizes, morphologies, zeta potentials, stabilities, entrapment efficiencies, loading efficiencies, and drug releases. Notably, the therapeutic effect of liposomes on an in vitro model of atherosclerosis has been validated. The data from this study show that targeted diagnosis and treatment of atherosclerosis is possible.
This work was supported by the National Key Research Program of China (2016YFA0100903), National Funds for Distinguished Young Scientists of China (81325009), National Nature Science Foundation of China (Nos. 81530058, 81570272, 81227901), and Beijing Nature Science Foundation (No. 7152131).
XH, MQX, WZ, and FC performed the research. XH and MQX analysed the data and wrote the paper. FC, WZ, SM, YDC, YBW, YZ, and TTG designed the research and reviewed the article. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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