The targeting mechanism of DHA ligand and its conjugate with Gemcitabine for the enhanced tumor therapy

Abstract

Docosahexaenoic acid (DHA), an omega-3 C22 natural fatty acid serving as a precursor for metabolic and biochemical pathways, was reported as a targeting ligand of anticancer drugs. However, its tumor targeting ability and mechanism has not been claimed. Here we hypothesized that the uptake of DHA by tumor cells is related to the phosphatidylethanolamine (PE) contents in cell membranes. Thus, in this manuscript, the tumor-targeting ability of DHA was initially demonstrated in vitro and in vivo on different tumor cell lines by labeling DHA with fluorescence dyes. Subsequently, the tumor targeting ability was then correlated with the contents of PE in cell membranes to study the uptake mechanism. Further, DHA was conjugated with anticancer drug gemcitabine (DHA-GEM) for targeted tumor therapy. Our results demonstrated that DHA exhibited high tumor targeting ability and PE is the main mediator, which confirmed our hypothesis. The DHA-GEM displayed enhanced therapeutic efficacy than that of GEM itself, indicating that DHA is a promising ligand for tumor targeted therapy.

Figure 1

A, synthetic scheme of DHA-Cypate, through modification of the carboxy group of DHA and Cypate using NHNH as reaction linkage B, the absorption spectra of DHA-Cypate, free DHA, free Cypate. C, fluorescence emission spectra of DHA-Cypate, free Cypate. D, mass spectrum of DHA-Cypate.

Figure 2

A, laser confocal fluorescence microscopy images of MCF-7, HepG-2, L02 cells incubated with DHA-RhB and free RhB B, mean fluorescence intensity of MCF-7, HepG-2, L02 cells incubated with DHA-RhB and free RhB. C, laser confocal fluorescence microscopy images of MCF-7, HepG-2 cells incubated with DHA-RhB, DHA block DHA-RhB, DHA combined with PE recover the DHA-RhB. Data are given as mean ± SD (n=5). *, P<0.05. DIC, differential interference contrast.

Figure 3

A, the value of the fluorescent polarization P and the viscosity η in MCF-7, HepG-2, L02 cells B, linear fit of the value of cell uptake ratio and viscosity, R²=0.924. Data are given as mean ± SD (n=5). *, P<0.05.

Figure 4

A, dynamics of Cyptae in MCF-7 bearing-nude mice, dynamics and tumor-targeting ability of DHA-Cypate in MCF-7, HepG-2 bearing nude mice, and DHA block DHA-Cypate in MCF-7, HepG-2 bearing nude mice B, tumor/normal tissue ratio (T/N ratio=[tumor signal -background signal]/[normal signal (muscle)-background signal]×100%) calculated from the ROIs at 2, 4, 6, 8, 12, 24hour postinjection of DHA-Cypate into MCF-7, HepG-2 bearing nude mice. C, NIR images of main organs excised from normal nude mice after intravenous injection of DHA-Cypate at 24h.

Figure 5

A, synthetic scheme of DHA-GEM, through modification of the carboxy group of DHA with the the primary amine group of gemcitabine B, mass spectrum of DHA-GEM.

Figure 6. In vitro and vivo antitumor efficacy of DHA-GEM on H22 cells and H22 tumor-bearing mice

A, Cell viability of H22 cells incubated with DHA-GEM and free gemcitabine. B, tumor volume of mice-bearing H22 tumors under different treatments (saline, free gemcitabine, or DHA-GEM, n=10/group). C, tumor weight and tumor picture of mice-bearing H22 tumors in different groups(saline, free gemcitabine, or DHA-GEM) on the 9th day after injection. D, body weights of mice bearing H22 tumors in different groups. E, the 20-day survival rates of mice after administration of saline, free gemcitabine, or DHA-GEM. F, hematoxylin and eosin-stained hearts, tumors, livers and kidneys of saline treated mice, gemcitabine-treated mice,or DHA-GEM–treated mice. Data are given as mean ± SD (n=10). ^**, P<0.01.

Figure 7. In vitro and vivo antitumor efficacy of DHA-GEM on MCF-7 cells and MCF-7 tumor-bearing nude mice

A, Cell viability of MCF-7 cells incubated with DHA-GEM, free DHA and free gemcitabine. B, tumor volume of mice-bearing MCF-7 tumors under different treatments (saline, free gemcitabine, or DHA-GEM, n=6/group). C, tumor weights and tumor picture of mice-bearing MCF-7 tumors in different groups (saline, free gemcitabine, or DHA-GEM) on the 24th day after injection. D, body weights of mice bearing MCF-7 tumors in different groups. Data are given as mean ± SD (n=6). ^**, P<0.01.