Targeting Anticancer Drug Delivery to Pancreatic Cancer Cells Using a Fucose-Bound Nanoparticle Approach

<div><p>Owing to its aggressiveness and the lack of effective therapies, pancreatic ductal adenocarcinoma has a dismal prognosis. New strategies to improve treatment and survival are therefore urgently required. Numerous fucosylated antigens in sera serve as tumor markers for cancer detection and evaluation of treatment efficacy. Increased expression of fucosyltransferases has also been reported for pancreatic cancer. These enzymes accelerate malignant transformation through fucosylation of sialylated precursors, suggesting a crucial requirement for fucose by pancreatic cancer cells. With this in mind, we developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specifically to cancer cells. L-fucose-bound liposomes containing Cy5.5 or Cisplatin were effectively delivered into CA19-9 expressing pancreatic cancer cells. Excess L-fucose decreased the efficiency of Cy5.5 introduction by L-fucose-bound liposomes, suggesting L-fucose-receptor-mediated delivery. Intravenously injected L-fucose-bound liposomes carrying Cisplatin were successfully delivered to pancreatic cancer cells, mediating efficient tumor growth inhibition as well as prolonging survival in mouse xenograft models. This modality represents a new strategy for pancreatic cancer cell-targeting therapy.</p> </div

Fuc-liposomes carrying Cisplatin suppressed tumor growth and prolonged survival of mice in the xenograft model.

<p>(<b>A, B</b>) Comparison of tumor growth suppression with Cisplatin, F0-Liposome-Cisplatin, and F50-Liposome-Cisplatin in AsPC-1-bearing mice. Cisplatin (2 mg/kg), F0-Liposome-Cisplatin (2 mg/kg), or F50-Liposome-Cisplatin solution (2 mg/Cisplatin/kg) was injected via the tail vein of AsPC-1-bearing mice twice a week. At 4, 8, 11, 15, 18<b>,</b> and 22 days after transplantation, tumor volumes were measured. Representative image of mice treated with Cisplatin (<b>B</b>). Results are expressed as the mean ± SD (n = 6). *P<0.01 compared with NT, Cisplatin, and F0. (<b>C</b>) Tumor tissue was prepared on day 22 after treatment. HE staining (upper panel) and TUNEL staining (lower panel) are presented. (<b>D</b>) Concentration of platinum in the tumor tissue measured by ICP. (<b>E</b>) Survival rate of the mice treated with Cisplatin alone, F0-Liposome-Ciplatin, and F50-Liposome-Ciplatin in the liver metastasis model using BxPC-3 cells. Statistical analysis was performed by generalized Wilcoxon test. *P<0.01 compared with NT, Cisplatin, and F0. (<b>F</b>) Localization of BxPC3-Luc cells in the orthotopic model detected using an IVIS imaging system after 3 weeks of treatment.</p

Production and physicochemical properties of L-fucose-bound liposomes.

<p>(<b>A)</b> Liposome preparation scheme showing sugar chains. HSA, BS³, Tris, and DTSSP denote the following, respectively: human serum albumin; bis(sulfosuccinimidyl) suberate; Tris(hydroxymethyl) aminomethane; 3,3-dithiobis (sulfosuccinimidylpropionate). (<b>B)</b> Electron microscopic image of L-fucose-bound liposome. Scale bar shows 50 nm. (<b>C, D)</b> Physicochemical characterization of Fuc-Liposome-Cy5.5. Average particle size <b>(C)</b> and zeta-potential <b>(D)</b> of liposomes that were prepared in water was determined by dynamic light scattering spectrophotometry.</p

Receptor-mediated uptake of Fuc-Liposomes by pancreatic cancer cells.

<p>(<b>A</b>) Incorporation of ¹⁴C-labeled-L-fucose in AsPC-1 cells. Cells were incubated in the presence or absence of excess L-fucose (excess cold) in the ¹⁴C-labeled-L-fucose-containing medium for the indicated time, then ¹⁴C-labeled-L-fucose incorporation was measured. (<b>B</b>) BxPC-3 cells were incubated with or without chroloquine for 24 hours, treated with F50-Liposome-Cy5.5 for 2 hours at 37°C, and then analyzed by flow cytometry. (<b>C, D</b>) ¹⁴C-labeled-L-fucose binding assay using AsPC-1 cells. Scatchard plot analysis revealed 3.25×10⁶ receptors/cell, a K_d of 28.74 nM, and a Bmax of 5.49 pmol/10⁶ cells. Methods are described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039545#s4" target="_blank">Materials and Methods</a></i>.</p

Pretreatment with D-mannose does not affect accumulation of F50-Liposome-Cy5.5.

<p>(<b>A</b>) Fuc-Liposome- or Liposome-Cy5.5 was administered via the tail vein (50 µl/mouse). The tumor regions of MIA PaCa-2, BxPC-3 and AsPC-1 cells (the back side of a bilateral flank lesion) in the mouse was observed and Cy5.5 accumulation was quantified using the IVIS imaging system at 96 hours after injection. D-mannose (1000-fold of the L-fucose) was injected simultaneously with liposome injection. (<b>B</b>) Total flux of the tumor and liver was calculated by using Living Image software according to the manufacturer’s instructions.</p

Effect of Fuc-Liposome-Cisplatin on the growth of pancreatic cancer cell lines.

<p>(<b>A, B</b>) Cells were treated with Fuc-Liposome containing Cisplatin for 2 hours, then washed and incubated for 72 hours. Viable cells were measured by WST assay.</p