Liposomal formulation of Galbanic acid improved therapeutic efficacy of pegylated liposomal Doxorubicin in mouse colon carcinoma

Abstract

Galbanic acid (Gba), a sesquiterpene coumarin, with strong antiangiogenic activity could serve as an excellent anti-cancer agent. However, Gba is a poor water-solube which hampered its clinical application. In this study, a pegylated liposomal Gba (PLGba) with HSPC/Cholesterol/mPEG₂₀₀₀-DSPE (56.2, 38.3, 5.3% molar ratio) was developed by the thin film hydration plus extrusion and calcium acetate gradient remote loading method, to address the issue of poor Gba solubility. Moreover, an integrin-targeting ligand (RGD peptide, cyclo[Arg-Gly-Asp-D-Tyr-Cys]) was post-inserted into liposomes in order to increase Gba cell delivery. Using fluorescently-labeled model liposomes, it was found that the targeting could improve the integrin-mediated cellular uptake of the liposomes in vitro in human umbilical vein endothelial cells (HUVECs), and in vivo as evidenced by chicken chorioallantoic membrane angiogenesis (CAM) model. It also could enrich the liposome accumulation in C26 tumor. Interestingly, co-treatment with PLGba and pegylated liposomal doxorubicin (PLD, also known as Doxil^®) had a synergistic and antagonistic antiproliferative effect on the C26 tumor cell line and the normal HUVEC, respectively. In C26 tumor bearing BALB/c mice, the PLGba and PLD combinatorial therapy improved the antitumor efficacy of the treatment as compared to those of single agents. This results have clear implications for cancer therapy.

Figure 1

MALDI-TOF mass spectroscopy analysis data for the RGD peptide (A), Mal-PEG₂₀₀₀-DSPE (B), and RGD-lipid (C) revealed the consumption of free DSPE-PEG2000-Mal in reaction and formation of the DSPE-PEG-RGD lipopeptide.

Figure 2

Transmission electron microscopy (TEM) images of negatively stained PLGba at magnification of ×25000 (A) and ×31500 (B).

Figure 3

Liposomal Gba content of PLGba and RGD-PLGba incubated at 37 °C in the PBS/FCS buffer for 96 hours. Data are presented as mean ± standard deviation (n = 3). Ns indicates non-significant (p > 0.5).

Figure 4

In vitro cellular binding affinity (A) and liposome-cell association (B) of the RGD-targeted and non-targeted fluorescently-labeled model liposomes in HUVECs at 4 °C and 37 °C, respectively. Data are shown as mean ± standard deviation of three independent experiments. * indicates significant differences (p < 0.05).

Figure 5

CAM vasculature development on day 12 of egg development treated with PLGba, RGD-PLGba, and Avastin (A) shows the stereomicroscope of the CAM vessels, (B) is the quantitative analysis of the chicken CAM using NIH ImageJ with the angiogenesis analyzer plugin. (C) and (D) are the numbers of vascular nodes and branches. Data are expressed as mean ± standard deviation (n = 6). * indicates significant difference as compared to control (p < 0.05).

Figure 6

In vivo fluorescence image and the related fluorescent intensity of tumor, tissues, and serum in BALB/c mice bearing C26 colon tumor injected with RGD-targeted and non-targeted fluorescently-labeled model liposomes. (A) and (B) are the real-time fluorescence images and the associated fluorescence intensity data at the mentioned time points. (C) indicates the concentration of the fluorescent dye (ng/g tissues) in tumor at 3 and 24 h post-injection. (D) and (E) are the tissues concentrations of the dye at 3 and 24 h post-injection, respectively; and (F) is the serum concentration of the dye within 48 h post-injection. Data are shown as mean ± standard deviation (n = 3). * indicates significant differences (p < 0.05).

Figure 7

In vivo therapeutic efficacy of f-Gba, Gba-liposomes, PLD and PLD + PLGba combination in C26 tumor-bearing mice treated with multiple doses of Gba (10 mg Gba/kg) and a single dose of PLD (10 mg Dox/kg). The Gba-liposomes did not change the body weight (A), but they delayed tumor growth up to day 22^th post-injection (B) and increased the lifespan, especially in mice treated with PLD + PLGba (C). Data are expressed as mean ± standard deviation (n = 5). ^#Indicates a significant difference between PLD and control group, ** indicates a significant difference between all treatment groups and the control group, and *indicates a significant difference between PLD + PLGba treatment group and all treatment groups (p < 0.05).