Cyclic RGD peptide-modified liposomal drug delivery system for targeted oral apatinib administration: enhanced cellular uptake and improved therapeutic effects

Abstract

Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin α_vβ₃ and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer.

Keywords: apatinib; cRGD; colorectal cancer; integrin αvβ3; targeted oral therapy.

Figure 1

Characteristic ¹H-NMR spectra. Abbreviations: ¹H-NMR, proton nuclear magnetic resonance; DSPE-PEG2000, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt; RGD, arginine–glycine–aspartic acid; cRGDfc, cyclo Arginine-Glycine-Asparticacid-D-Phenylalanine-Cycstine; Mal, maleimide.

Figure 2

(A) The particle size and zeta potential of blank Lipo-PEG, cRGD-Lipo-PEG, Apa-loaded Lipo-PEG, and Apa-loaded cRGD-Lipo-PEG liposomes, and (B) size distribution graph of Apa-encapsulating Lipo-PEG and cRGD-Lipo-PEG. Abbreviations: PDI, polydispersity index; PEG, polyethylene glycol; cRGD, cyclic arginylglycylaspartic acid; Apa, apatinib; Lipo, liposomes.

Figure 3

The variations in particle sizes and zeta potentials of (A) Lipo-PEG/Apa and (B) cRGD-Lipo-PEG/Apa over 12 days. Abbreviations: PDI, polydispersity index; PEG, polyethylene glycol; Apa, apatinib; Lipo, liposomes; cRGD, cyclic arginylglycylaspartic acid.

Figure 4

In vitro study of Apa release for free Apa, Lipo-PEG/Apa, and cRGD-Lipo-PEG/Apa in PBS at (A) pH =7.4 and (B) pH =5.6 over 72 hours. Abbreviations: PEG, polyethylene glycol; Apa, apatinib; Lipo, liposomes; cRGD, cyclic arginylglycylaspartic acid; PBS, phosphate-buffered saline.

Figure 5

Expression analysis of β₃ integrins in (A) HCT116 cells (two samples) and 293T cells (two samples) and (B) liver, kidney and HCT116 tumor are from the same animal.

Figure 6

Fluorescence and flow cytometry experiments in HCT116 cells. Notes: (A) Fluorescence microscope images showing the cellular uptake in HCT116 cells. Magnification ×20. (B) Flow cytometry charts showing the cellular uptake in HCT116 cells. (C) Mean fluorescence intensity and cellular uptake percentage as determined by flow cytometry experiments. (D) Fluorescence microscope images showing the cellular uptake in 293T cells. Magnification ×20. (E) Flow cytometry charts showing the cellular uptake in 293T cells. (F) Mean fluorescence intensity and cellular uptake percentage as determined by flow cytometry experiments. Abbreviations: cRGD, cyclic arginylglycylaspartic acid; PEG, polyethylene glycol; Lipo, liposomes; C6, coumarin 6.

Figure 6

Fluorescence and flow cytometry experiments in HCT116 cells. Notes: (A) Fluorescence microscope images showing the cellular uptake in HCT116 cells. Magnification ×20. (B) Flow cytometry charts showing the cellular uptake in HCT116 cells. (C) Mean fluorescence intensity and cellular uptake percentage as determined by flow cytometry experiments. (D) Fluorescence microscope images showing the cellular uptake in 293T cells. Magnification ×20. (E) Flow cytometry charts showing the cellular uptake in 293T cells. (F) Mean fluorescence intensity and cellular uptake percentage as determined by flow cytometry experiments. Abbreviations: cRGD, cyclic arginylglycylaspartic acid; PEG, polyethylene glycol; Lipo, liposomes; C6, coumarin 6.

Figure 7

The cytotoxicity study of (A) free apatinib, (B) blank liposomes, as well as (C) their apatinib-loaded liposomes in HCT116 cells. Abbreviations: IC₅₀, half-maximal inhibitory concentration; cRGD, cyclic arginylglycylaspartic acid; PEG, polyethylene glycol; Lipo, liposomes.

Figure 8

(A) The apoptosis assay in HCT116 cells after treatment with apatinib-loaded liposomes and free apatinib for 24 h at a concentration of 2 µM; (B) the apoptotic ratio in the three groups. Abbreviations: Apa, apatinib; cRGD, cyclic arginylglycylaspartic acid; PEG, polyethylene glycol; Lipo, liposomes.

Figure 9

The representative images and the detailed biodistribution data in vivo (A and C) and ex vivo (B and D) of HCT116 tumor-bearing models after intravenous injection of free DiR and DiR-labeled liposomes. Notes: The red arrows in (A) indicate the location of HCT116 tumors. *P<0.05; **P<0.01. Abbreviations: DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide; cRGD, cyclic arginylglycylaspartic acid; Lipo, liposomes; PEG, polyethylene glycol.

Figure 10

The representative images and the detailed biodistribution data (A and C) in vivo and (B and D) ex vivo of HCT116 tumor-bearing models after intragastric administration of free DiR and DiR-labeled liposomes. Notes: The red arrows in (A) indicate the location of HCT116 tumors. *P<0.05; **P<0.01. Abbreviations: DiR, 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide; Lipo, liposomes; PEG, polyethylene glycol; cRGD, cyclic arginylglycylaspartic acid.

Figure 11

The antitumor activity of free apatinib and cRGD-Lipo-PEG/Apa on HCT116 tumor-bearing nude mice. Notes: (A) Tumor volume growth curves. (B) The weights of tumors at the end of different treatments. (C) The photographs of tumors at the end of different treatments. (D) The organs’ coefficients at the end of different treatments. (E) Body weight variations of mice during the treatment. Abbreviations: cRGD, cyclic arginylglycylaspartic acid; Lipo, liposomes; PEG, polyethylene glycol; Apa, apatinib; PBS, phosphate-buffered saline.

Figure 11

The antitumor activity of free apatinib and cRGD-Lipo-PEG/Apa on HCT116 tumor-bearing nude mice. Notes: (A) Tumor volume growth curves. (B) The weights of tumors at the end of different treatments. (C) The photographs of tumors at the end of different treatments. (D) The organs’ coefficients at the end of different treatments. (E) Body weight variations of mice during the treatment. Abbreviations: cRGD, cyclic arginylglycylaspartic acid; Lipo, liposomes; PEG, polyethylene glycol; Apa, apatinib; PBS, phosphate-buffered saline.

Figure 12

H–E staining of major organs after treatment. Note: Magnification ×100. Abbreviations: H–E, hematoxylin and eosin; Lipo, liposomes; PBS, phosphate-buffered saline.

Scheme 1

Schematic illustration of cRGD-modified liposomes (cRGD-Lipo-PEG). Notes: The liposomes were internalized into tumor cells via the cRGD ligand, achieving sufficient cellular uptake efficacy; moreover, they were targeted to solid tumors actively and passively in vivo. Abbreviations: SPC, soybean phosphatidylcholine; cRGD, cyclic arginylglycylaspartic acid; Lipo, liposomes; PEG, polyethylene glycol; DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt.