Inhibition of tumor metastasis by targeted daunorubicin and dioscin codelivery liposomes modified with PFV for the treatment of non-small-cell lung cancer

Abstract

Background: Chemotherapy for non-small-cell lung cancer (NSCLC) still leads to unsatisfactory clinical prognosis because of poor active targeting and tumor metastasis. Purpose: The objective of this study was to construct a kind of PFV peptide modified targeted daunorubicin and dioscin codelivery liposomes, which could enhance tumor targeting and inhibit tumor cell metastasis. Methods and results: Targeted daunorubicin and dioscin codelivery liposomes were prepared by film dispersion and the ammonium sulfate gradient method. With the ideal physicochemical properties, targeted daunorubicin and dioscin codelivery liposomes exhibited enhanced cellular uptake and showed strong cytotoxicity to tumor cells. The encapsulation of dioscin increased the inhibitory effects of daunorubicin on A549 cells, vasculogenic mimicry (VM) channels and tumor metastasis. The enhanced antimetastatic mechanism of the targeted liposomes was attributed to the downregulation of matrix metalloproteinase-2 (MMP-2), vascular endothelial cadherin (VE-Cad), transforming growth factor-β1 (TGF-β1) and hypoxia inducible factor-1α (HIF-1α). Meanwhile, the targeted daunorubicin and dioscin codelivery liposomes exhibited significant antitumor effects in tumor-bearing mice. H&E staining, immunohistochemistry with Ki-67 and TUNEL assay also showed the promoted antitumor activity of the targeted liposomes. Conclusion: Targeted daunorubicin and dioscin codelivery liposomes may provide an effective strategy for the treatment of NSCLC.

Keywords: cell penetrating peptides; daunorubicin; dioscin; liposomes; non-small-cell lung cancer; tumor metastasis.

Figure 1

Schematic illustration of strategy of inhibiting tumor metastasis by targeted daunorubicin and dioscin codelivery liposomes. Abbreviations: PFV, PFVYLI; DSPE-PEG₂₀₀₀, polyethylene glycol-distearoyl phosphatidylethanolamine; EPR effect, enhanced permeability and retention effect; VE-Cad, vascular endothelial cadherin; TGF-β1, transforming growth factor-β1; HIF-1α, hypoxia inducible factor-1α; MMP-2, matrix metalloproteinase-2.

Figure 2

Characterization of liposomes. (A1) MALDI-TOF-MS spectrum of DSPE-PEG₂₀₀₀-COOH, (A2) MALDI-TOF-MS spectrum of DSPE-PEG₂₀₀₀-PFV, (B) TEM image of targeted daunorubicin and dioscin codelivery liposomes, (C) In vitro cumulative release profile of daunorubicin from varying liposomal formulations. Data are presented as mean ± SD (n=3). △, vs Daunorubicin liposomes; #, vs Daunorubicin and dioscin codelivery liposomes. p<0.05, (D1) AFM image of targeted daunorubicin and dioscin codelivery liposomes, (D2) 3D structure of (D1). Abbreviations: PFV, PFVYLI; DSPE-PEG₂₀₀₀-COOH, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [carboxy (polyethylene glycol)-2000]; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; TEM, transmission electron microscope; AFM, atomic force microscope.

Figure 3

Cellular uptake and targeting effects after incubation with the varying formulations. (A1) Cellular uptake of A549 cells treated with targeted daunorubicin liposomes consist varying content of PFV for 0.5 h, (A2) Cellular uptake of A549 cells treated with targeted daunorubicin liposomes consist varying content of PFV for 1 h, (A3) Cellular uptake of A549 cells treated with targeted daunorubicin liposomes consist varying content of PFV for 2 h, (A4) Cellular uptake of A549 cells treated with targeted daunorubicin liposomes consist varying content of PFV for 3 h, (A5) Fluorescence intensity of (A1), (A6) Fluorescence intensity of (A2), (A7) Fluorescence intensity of (A3), (A8) Fluorescence intensity of (A4). a, EPC:DSPE-PEG₂₀₀₀-PFV=100:0; b, EPC:DSPE-PEG₂₀₀₀-PFV=100:1; c, EPC:DSPE-PEG₂₀₀₀-PFV=100:2; d, EPC:DSPE-PEG₂₀₀₀-PFV=100:4. Data are presented as mean ± SD (n=3). I, vs a; II, vs b. p<0.05. (B1) Cellular uptake of A549 cells treated with varying liposomal formulations or free drug, (B2) Fluorescence intensity of (B1). 1, Daunorubicin liposomes; 2, Daunorubicin and dioscin codelivery liposomes; 3, Targeted daunorubicin and dioscin codelivery liposomes; 4, Free daunorubicin. △, vs Daunorubicin liposomes; #, vs Daunorubicin and dioscin codelivery liposomes. p<0.05. (C) Fluorescence microscopy images of A549 cells incubated with the varying formulations. Abbreviations: EPC, egg yolk phosphatidylcholine; PFV, PFVYLI.

Figure 4

Inhibitory effects on A549 cells after treatments with the varying formulations. (A) Inhibitory effects of free drugs. I, vs Free dioscin; II, vs Free daunorubicin; III, vs Free daunorubicin: free dioscin=2:1; IV, vs Free daunorubicin: free dioscin=1:1; (B) Inhibitory effects of liposomal formulations. 1, vs Blank liposomes; 2, vs Dioscin liposomes; 3, vs Daunorubicin liposomes; 4, vs Daunorubicin and dioscin codelivery liposomes. Data are presented as mean ± SD (n=6). p<0.05.

Figure 5

Inhibition of VM formation, wound healing and tumor migration. (A) Inhibition of VM formation, (B) Blocking effects on wound healing, (C) Blocking effects on tumor migration. Abbreviation: VM, vasculogenic mimicry.

Figure 6

Regulating effects on metastasis-related proteins. (A) MMP-2 protein expression ratio, (B) VE-Cad protein expression ratio, (C) TGF-β1 protein expression ratio, (D) HIF-1α protein expression ratio. Data are presented as mean ± SD (n=3). a. Blank control; b. Dioscin liposomes; c. Daunorubicin liposomes; d. Daunorubicin and dioscin codelivery liposomes; e. Targeted daunorubicin and dioscin codelivery liposomes. 1, vs a; 2, vs b; 3, vs c; 4, vs d. p<0.05. Abbreviations: MMP-2, matrix metalloproteinase-2; VE-Cad, vascular endothelial cadherin; TGF-β1, transforming growth factor-β1; HIF-1α, hypoxia inducible factor-1α.

Figure 7

Apoptotic effects to A549 cells. (A) Apoptosis determined by flow cytometry, (B) The proportion of apoptosis after varying liposomal formulations treatment. Data are presented as mean ± SD (n=3). a. Blank control; b. Dioscin liposomes; c. Daunorubicin liposomes; d. Daunorubicin and dioscin codelivery liposomes; e. Targeted daunorubicin and dioscin codelivery liposomes. 1, vs a; 2, vs b; 3, vs c; 4, vs d. p<0.05. (C) Fluorescence microscopy images of intracellular ROS level in A549 cells. Abbreviation: ROS, reactive oxygen species.

Figure 8

In vivo imaging in mice. Abbreviation: DiR, 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide.

Figure 9

In vivo antitumor efficacy in tumor-bearing mice. (A) Body weight changes, (B) Tumor volume changes. △, vs Blank control, p<0.05, (C) H&E staining assay, (D) Immunohistochemistry staining of the proliferation marker Ki-67 in tumor tissues. a, Blank control; b, Free daunorubicin; c, Dioscin liposomes; d, Daunorubicin liposomes; e, Daunorubicin and dioscin codelivery liposomes; f, Targeted daunorubicin and dioscin codelivery liposomes, (E) TUNEL assay for apoptotic cells.