Figures & data
Figure 1 The preparation of iRGD-SSL-DOX.
Note: (A)The synthesis of iRGD-PEG-DSPE and (B) the preparation of iRGD-SSL-DOX.
Abbreviations: iRGD-SSL-DOX, doxorubicin-loaded iRGD-modified sterically-stabilized liposome; iRGD, tumor-homing peptide; SSL, sterically-stabilized liposome; DOX, doxorubicin; DSPE-PEG-MAL, 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Maleimide(polyethylene-Glycol)-2000; PEG, polyethylene glycol; DSPE, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; EPC, egg phosphatidylcholine.
Table 1 The characteristics of iRGD-SSL-DOX (n = 3)
Figure 2 The typical particle size and distribution of SSL, SSL-DOX, and iRGD-SSL-DOX.
Notes: The typical particle size and distribution of (A) SSL, (B) SSL-DOX, and (C) iRGD-SSL-DOX.
Abbreviations: SSL, sterically-stabilized liposome; SSL-DOX, sterically-stabilized liposome containing DOX; DOX, doxorubicin; iRGD-SSL-DOX, DOX-loaded iRGD-modified SSL; iRGD, tumor-homing peptide.
Figure 3 The leakage of DOX from iRGD-SSL-DOX.
Notes: The medium was PBS (pH = 7.4). The temperature was controlled at 37°C. Each datum exhibits the mean ± SD (n = 3).
Abbreviations: DOX, doxorubicin; iRGD-SSL-DOX, DOX-loaded iRGD-modified SSL; SSL, sterically-stabilized liposome; PBS, phosphate buffered saline; SD, standard deviation.
Figure 4 The flow cytometric measurement of coumarin-6 uptake from SSL-coumarin-6 or iRGD-SSL-coumarin-6 by B16-F10 cells and MCF-7 cells, with or without trypsin treatment, at the 2 hours incubation time point.
Notes: (A) B16-F10 cells, without trypsin treatment groups. (B) B16-F10 cells, with trypsin treatment groups. (C) MCF-7 cells, without trypsin treatment groups. (D) MCF-7 cells, with trypsin treatment groups. Black exhibits control, blue exhibits incubation with SSL-coumarin-6, pink exhibits incubation with iRGD-SSL-coumarin-6, and red exhibits coumarin-6.
Abbreviations: SSL, sterically-stabilized liposome; iRGD, tumor-homing peptide; MCF-7, breast cancer cell line.
Figure 5 The confocal microscopy images of B16-F10 cells incubated with (A) SSL-coumarin-6, (B) iRGD-SSL-coumarin-6 or (C) free coumarin-6, for 2 hours at 37°C. Fluorescence of coumarin-6 exhibits green; fluorescence of Hoechst 33258 exhibits blue.
Abbreviations: iRGD, tumor-homing peptide; SSL, sterically-stabilized liposome.
Figure 6 In vivo image of biodistribution of iRGD-SSL-DiR in B16-F10 tumor-bearing nude mice.
Note: In vivo whole body imaging of B16-F10 tumor-bearing nude mice after IV administered physiological saline (as control), SSL-DiR, and iRGD-SSL-DiR, respectively.
Abbreviations: SSL, sterically-stabilized liposome; DiR, carbocyanine dye 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide; iRGD, tumor-homing peptide; IV, intravenous.
Figure 7 The localization of iRGD-SSL-DiI in tumor section.
Notes: B16-F10 tumor-bearing nude mice were IV administered with SSL-DiI or iRGD-SSL-DiI at a dose of 1200 ng/animal. At the 3-hour time point, the mice were sacrificed, and the tumors were harvested. The tumor sections were immune-stained and examined for fluorescence. Images in cluster represent (A) SSL-DiI treatment group, and (B) iRGD-SSL-DiI treatment group. Nuclei were counterstained with Hoechst 33258 (blue; A1 and B1). Tumor blood vessels represented a green fluorescence (antiCD31, green; A2 and B2). DiI represented a red fluorescence (red; A3 and B3). Merge (A4 and B4).
Abbreviations: iRGD, tumor-homing peptide; SSL, sterically-stabilized liposome; DiI, fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; IV, intravenous.
Figure 8 In vivo antitumor activity of iRGD-SSL-DOX.
Notes: C57BL/6 mice were inoculated SC with B16-F10 cells and treated with physiological saline, SSL-DOX (3 mg/kg, IV, q3d), iRGD-SSL-DOX (3 mg/kg, IV, q3d). The formulations were given via the tail vein for all administrations. The tumors were measured with a caliper twice per week throughout the study. **P < 0.01 versus physiological saline as control; ††P < 0.01 versus SSL-DOX treatment group. ↑, administration.
Abbreviations: iRGD-SSL-DOX, doxorubicin-loaded iRGD-modified sterically-stabilized liposome; iRGD, tumor-homing peptide; SSL, sterically-stabilized liposome; DOX, doxorubicin; SC, subcutaneous; IV, intravenous; q3d, every 3 days for three doses.
Figure 9 Kaplan–Meier survival curves of B16-F10 tumor-bearing C57BL/6 mice treated with iRGD-SSL-DOX.
Notes: Kaplan–Meier survival curves of B16-F10 tumor-bearing C57BL/6 mice treated with physiological saline (black), SSL-DOX (3 mg/kg, IV, q3d) (brown), iRGD-SSL-DOX (3 mg/kg, IV, q3d) (green). Results indicated that the iRGD-SSL-DOX (43.5 days) significantly improved the median survival time of mice as compared with that treated with SSL-DOX (33 days, P < 0.01) and physiological saline (27 days, P < 0.01), respectively.
Abbreviations: iRGD-SSL-DOX, doxorubicin-loaded iRGD-modified sterically-stabilized liposome; iRGD, tumor-homing peptide; SSL, sterically-stabilized liposome; DOX, doxorubicin; IV, intravenous; q3d, every 3 days for three doses.
Figure 10 The detailed scheme of tumor-targeting and tumor-penetrating effect of iRGD-SSL-DOX.
Note: The RGD motif in iRGD-SSL-DOX mediates binding to αν integrins firstly on tumor cells followed by a proteolytic cleavage, exposing a binding motif of CRGDK sequences for NRP-1, which can then mediate penetration into tumor cells.
Abbreviations: iRGD-SSL-DOX, doxorubicin-loaded iRGD-modified sterically-stabilized liposome; iRGD, tumor-homing peptide; SSL, sterically-stabilized liposome; DOX, doxorubicin; CRGDK, Cys-Arg-Gly-Asp-Lys.
