Therapeutic effect of apatinib-loaded nanoparticles on diabetes-induced retinal vascular leakage

Abstract

Apatinib, a novel and selective inhibitor of vascular endothelial growth factor (VEGF) receptor 2, has been demonstrated recently to exhibit anticancer efficacy by inhibiting the VEGF signaling pathway. Given the importance of VEGF in retinal vascular leakage, the present study was designed to investigate whether apatinib-loaded polymeric nanoparticles inhibit VEGF-mediated retinal vascular hyperpermeability and block diabetes-induced retinal vascular leakage. For the delivery of water-insoluble apatinib, the drug was encapsulated in nanoparticles composed of human serum albumin (HSA)-conjugated polyethylene glycol (PEG). In vitro paracellular permeability and transendothelial electric resistance assays showed that apatinib-loaded HSA-PEG (Apa-HSA-PEG) nanoparticles significantly inhibited VEGF-induced endothelial hyperpermeability in human retinal microvascular endothelial cells. In addition, they substantially reduced the VEGF-induced junctional loss and internalization of vascular endothelial-cadherin, a major component of endothelial junction complexes. In vivo intravitreal injection of Apa-HSA-PEG nanoparticles in mice blocked VEGF-induced retinal vascular leakage. These in vitro and in vivo data indicated that Apa-HSA-PEG nanoparticles efficiently blocked VEGF-induced breakdown of the blood-retinal barrier. In vivo experiments with streptozotocin-induced diabetic mice showed that an intravitreal injection of Apa-HSA-PEG nanoparticles substantially inhibited diabetes-induced retinal vascular leakage. These results demonstrated, for the first time, that apatinib-loaded nanoparticles may be a promising therapeutic agent for the prevention and treatment of diabetes-induced retinal vascular disorders.

Keywords: permeability; retinal vascular endothelial cells; vascular endothelial growth factor.

Figures

Figure 1

Preparation and characterization of Apa-HSA-PEG nanoparticles. Notes: (A) Schematic diagram of the preparation of Apa-HSA-PEG nanoparticles. (B) TEM images and (C) particle size distribution of Apa-HSA-PEG nanoparticles. All scale bars =500 µm. Abbreviations: Apa-HSA-PEG, apatinib-loaded human serum albumin-conjugated polyethylene glycol; TEM, transmission electron microscope; NHS, N-hydroxysuccinimide; RT, room temperature; THF, tetrahydrofuran.

Figure 2

Apa-HSA-PEG nanoparticles block VEGF-induced hyperpermeability in HRMECs. Notes: (A) Cytotoxicity of Apa-HSA-PEG nanoparticles and apatinib was evaluated using the Cell Counting Kit-8 assay (n=6). HRMECs were incubated with apatinib (1 and 5 µM) or Apa-HSA-PEG nanoparticles (960 and 4.8 µg corresponding to 1 and 5 µM apatinib, respectively) for 1–2 days. (B and C) Endothelial permeability was evaluated by measuring the (B) passage of FITC-dextran and (C) TEER in a HRMEC monolayer. HRMECs pretreated with apatinib (1 µM) or Apa-HSA-PEG nanoparticles (6.1 µg corresponding to 1 µM apatinib), and untreated cells were stimulated with rhVEGF (50 ng/mL). (B) FITC-dextran permeability is expressed as the fold change ± SEM with respect to the PBS control (*P<0.05 vs PBS control, #P<0.05 vs VEGF, n=6). (C) In the TEER experiment, specified reagents were added to the upper chamber at time zero, and serial changes in electrical resistance were measured 30 and 60 minutes later. Electrical resistance is expressed as the fold change ± SEM relative to the PBS control at each time point (*P<0.05 vs PBS control, #P<0.05 vs VEGF, n=6). Abbreviations: Apa-HSA-PEG, apatinib-loaded human serum albumin-conjugated polyethylene glycol; VEGF, vascular endothelial growth factor; HRMECs, human retinal microvascular endothelial cells; FITC, fluorescein isothiocyanate; TEER, transendothelial electrical resistance; rhVEGF, recombinant human VEGF; SEM, standard error of the mean; PBS, phosphate-buffered saline; NS, nonsignificant.

Figure 3

Apa-HSA-PEG nanoparticles prevent VEGF-induced internalization of VE-cadherin. Notes: (A) Representative immunofluorescent images of VE-cadherin. (B) Quantification of internalized VE-cadherin in HRMECs (means ± SEM, *P<0.05 vs PBS, #P<0.05 vs VEGF, n=4). Cells pretreated with Apa-HSA-PEG nanoparticles (6.1 µg corresponding to 1 µM apatinib) or PBS were stimulated with rhVEGF (50 ng/mL). Arrowheads in the no wash image indicate the disappearance of VE-cadherin (green) at endothelial junctions that were stained positively for anti-ZO1 IgGs (red). Arrowheads in the acid wash image indicate internalized VE-cadherin (green) in endosomes that were stained positively for EEA1 (red). Nuclei are shown in blue (DAPI). Scale bars =25 µm. Abbreviations: Apa-HSA-PEG, apatinib-loaded human serum albumin-conjugated polyethylene glycol; EEA1, early endosome antigen 1; VEGF, vascular endothelial growth factor; VE, vascular endothelial; HRMECs, human retinal microvascular endothelial cells; SEM, standard error of the mean; PBS, phosphate-buffered saline; rhVEGF, recombinant human VEGF; DAPI, 4′,6-diamidino-2-phenylindole; VE-cad, VE-cadherin.

Figure 4

Apa-HSA-PEG nanoparticles block VEGF-induced retinal vascular leakage in vivo. Notes: (A) Representative images of FITC-dextran-perfused retinal whole mounts. (B) Quantitative analysis of extravasated EB dye in the retinal tissues after intravitreal injection of rhVEGF (100 ng) and/or Apa-HSA-PEG nanoparticles (580 ng). Vascular leakage of EB dye in treated eyes was normalized relative to that in each contralateral control eye (means ± SEM, *P<0.05 vs PBS control, #P<0.05 vs VEGF, n=5). Scale bar =200 µm. Abbreviations: Apa-HSA-PEG, apatinib-loaded human serum albumin-conjugated polyethylene glycol; VEGF, vascular endothelial growth factor; FITC, fluorescein isothiocyanate; EB, Evans Blue; rhVEGF, recombinant human VEGF; SEM, standard error of the mean; PBS, phosphate-buffered saline.

Figure 5

Intravitreal injection of Apa-HSA-PEG nanoparticles inhibits diabetes-induced retinal vascular leakage in STZ-induced diabetic mice. Notes: (A) STZ-induced diabetic mice received an intravitreal injection of Apa-HSA-PEG nanoparticles (61 ng for the low-dose group [Low], 610 ng for the high-dose group [High]). An equal volume of PBS was injected into the contralateral eye as a control. One day after injection, retinal vascular leakage of EB dye was measured and expressed relative to that in each contralateral control eye (means ± SEM, **P<0.01 vs contralateral PBS control, n=8). (B) Blood glucose and body weights of mice used in this experiment were measured before (Nondiabetes) and 2 weeks after (Diabetes) the initial STZ injection (*P<0.05 vs Nondiabetes, n=8). Abbreviations: Apa-HSA-PEG, apatinib-loaded human serum albumin-conjugated polyethylene glycol; STZ, streptozotocin; PBS, phosphate-buffered saline; EB, Evans Blue; SEM, standard error of the mean.