Long-Circulating Curcumin-Loaded Liposome Formulations with High Incorporation Efficiency, Stability and Anticancer Activity towards Pancreatic Adenocarcinoma Cell Lines In Vitro

Abstract

The incorporation of hydrophobic drugs into liposomes improve their bioavailability and leads to increased stability and anticancer activity, along with decreased drug toxicity. Curcumin (Cur) is a natural polyphenol compound with a potent anticancer activity in pancreatic adenocarcinoma (PA). In the present study, different types of Cur-loaded liposomal formulations were prepared and characterized in terms of size, shape, zeta potential, optimal drug-to-lipid ratio and stability at 4°C, 37°C; and in human plasma in vitro. The best formulation in terms of these parameters was PEGylated, cholesterol-free formulation based upon hydrogenated soya PC (HSPC:DSPE-PEG2000:Cur, termed H5), which had a 0.05/10 molar ratio of drug-to-lipid, was found to be stable and had a 96% Cur incorporation efficiency. All Cur-loaded liposomal formulations had potent anticancer activity on the PA cancer cell lines AsPC-1 and BxPC-3, and were less toxic to a normal cell line (NHDF). Furthermore, apoptosis-induction induced by Cur in PA cells was associated with morphological changes including cell shrinkage, cytoplasmic blebbing, irregularity in shape and the externalization of cell membrane phosphatidylserine, which was preceded by an increase in intracellular reactive oxygen species (ROS) generation and caspase 3/7 activation. Because the liposomal formulations tested here, especially the H5 variant which exhibited slow release of the Cur in the human plasma test, the formulation may be stable enough to facilitate the accumulation of pharmacologically active amounts of Cur in target cancer tissue by EPR. Therefore, our formulations could serve as a promising therapeutic approach for pancreatic cancer and other cancers.

Fig 1. Appearance of liposomal formulations.

(A) Cur-loaded liposomes (left) and free liposomes (right). (B) TEM images of Cur-loaded liposomes (H5 formulation). Scale bar represents 200 nm.

Fig 2

Incorporation efficiency (IE) of Cur in different liposome-types (A). Comparison of the incorporation efficiency between C-HSPC-Cho-L (HCh10) and C-SPC-Cho-L (SCh10) formulations when 10% molar of Cur was added (B). The results are shown as the mean ± SD of three replicates.

Fig 3. Stability of Cur-loaded liposomes over different storage times and temperature.

Comparison of the stability of H5, H10, SCh5 and SCh10 formulations at 4°C (A) and at 37°C (B). The results are shown as the mean ± SD of three replicates.

Fig 4. The plasma stability of curcumin-loaded liposomes for the H5, H10, SCh5 and SCh10 formulations.

The results are shown as the mean ± SD of three replicates.

Fig 5. Cytotoxicity of free Cur, free liposomes, H5, H10, SCh5 and SCh10 formulations on AsPC-1, BxPC-3 and NDHF cells using MTT and ATP assays for 72 hours.

(A) AsPC-1 and MTT, (B) AsPC-1 and ATP, (C) BxPC-3 and MTT, (D) BxPC-3 and ATP; and (E) NDHF and MTT. The data are shown as the mean ± SD of three independent replicates. Statistical analysis by unpaired student t-test: *p<0.05; **p<0.001.

Fig 6. Effect of Cur treatment on ROS generation.

Intracellular ROS concentration was determined by treating AsPC-1 (A) and BxPC-3 (B) cells with free Curcumin and Curcumin in the H5 formulation, with Cur used at the effective IC50 concentration at time intervals of 1, 3, 6 and 24 hours, respectively. The data are shown as the mean ± SD of three independent replicates. Statistical analysis by unpaired student t-test: *p<0.05; **p<0.001.

Fig 7. Effect of curcumin treatment on caspase-3/7 activity and morphological changes in AsPC-1 cells.

AsPC-1 cells were treated with free curcumin and the H5 formulation containing curcumin, with the effective curcumin concentration equivalent to the IC50 in both cases. Caspase-3/7 activity (A), with morphological changes in real-time using phase-contrast and green fluorescence images showing apoptosis (B). The data are shown as the mean ± SD of three independent replicates. Statistical analysis by unpaired student t-test: *p<0.05; **p<0.001. Scale bar represents 300 μm.

Fig 8. Effect of curcumin treatment on caspase-3/7 activity and morphological changes in BxPC-3 cells.

BxPC-3 cells were treated with free curcumin and the H5 formulation containing curcumin, with the effective curcumin concentration equivalent to the IC50 in both cases. Caspase-3/7 activity (A), with morphological changes in real-time using phase-contrast and green fluorescence images showing apoptosis (B). The data are shown as the mean ± SD of three independent replicates. Statistical analysis by unpaired student t-test: *p<0.05; **p<0.001. Scale bar: represents 300 μm.

Fig 9. Detection of apoptotic cells using annexin V-FITC and PI double-staining.

AsPC-1 cells (A) and BxPC-3 cells (B) were treated with curcumin in the H5 formulation at a concentration equivalent to the IC50 of curcumin for 72 hours. Early (annexin V-FITC staining) and late (PI staining) apoptotic stages are shown. Scale bar represents 50 μm.