Purpose: Irinotecan is approved to be the first and second line chemotherapeutic drug and used to fight against various solid tumors. However, after repeated treatments, the curative effect of the irinotecan may be gradually hindered due to drug resistance. Therefore, strategies targeting to non-overlapping mechanisms may offer potential opportunities for those cancer treatments. In this study, a PEGylated dual-effect irinotecan liposome, PL-IRT-Ce6, which combined chemotherapy with photodynamic therapy (PDT) was developed to overcome the obstacle of drug resistance. Meanwhile, the therapeutic effect on the drug-resistant cell lines was also examined by another PEGylated dual-effect doxorubicin liposome, PL-Dox-Ce6.
Methods: Irinotecan (IRT) and chlorin e6 (Ce6) were co-encapsulated in the liposome (named as PL-IRT-Ce6) (Figure 1); similarly, doxorubicin (Dox) and Ce6 were co-loaded in the liposome (PL-Dox-Ce6). Both PL-IRT-Ce6 and PL-Dox-Ce6 were produced by the ammonium gradient method with Ce6 entrapped in the lipid bilayer while IRT or Dox encapsulated inside the liposome core. The stability of the liposome was monitored in 80% fetal bovine serum (FBS) at 37℃ using a water bath kept in the dark. MIA PaCa-2, AsPC-1, and MIA PaCa-2 transfected with ABCG2 transporter plasmid, MIA PaCa-2/ABCG2, were used to examine the therapeutic effect of PL-IRT-Ce6 and PL-Dox-Ce6. To verify the ability of resistance within different cell lines, drug sensitivity experiment was conducted and ABCG2 mRNA expression level was examined. In vitro cytotoxicity of PL-IRT-Ce6 and PL-Dox-Ce6 was studied by MTT assay, and the intracellular drug accumulation was evaluated by the cell uptake experiment. In both of the experiments, PDT was proceeded by the light irradiation using a 662 nm diode laser.
Results: In PL-IRT-Ce6, approximately 100% entrapment efficiency of IRT and 50% entrapment efficiency of Ce6 could be achieved. The average particle size of PL-IRT-Ce6 was around 150 nm. The lipid recovery of these liposomes was about 70% (Table 1). In the serum stability experiment, PL-IRT-Ce6 and PL-IRT were not very stable in serum. After one hour exposure in serum, only 80% of IRT remained in PL-IRT-Ce6. After two hours, the IRT remained in PL-IRT-Ce6 was down to 50%. On the other hand, PL-Dox-Ce6 was more stable compared to PL-IRT-Ce6 in the serum. This result could be explained by difference in liposome morphology of PL-IRT-Ce6 and PL-Dox-Ce6. In PL-Dox-Ce6, Dox formed crystals in the liposome, while IRT formed precipitates rather than crystals in PL-IRT-Ce6. As for the verification of drug resistance level, AsPC-1 showed higher viability against IRT and Dox than MIA PaCa-2, demonstrating that AsPC-1 was more resistant than MIA PaCa-2. Correspondingly, ABCG2 mRNA expression level of AsPC-1 was greater than MIA PaCa-2. In addition, MIA PaCa-2/ABCG2 exhibited much more ABCG2 mRNA than MIA PaCa-2, serving as an ABCG2 resistant cell line. In the cytotoxicity assay, both of the dual-effect liposomes showed improved toxicity after PDT treatment (Figure 2). Furthermore, the result of the cell uptake experiment demonstrated that dual-effect liposomes with PDT combination treatment could enhance the intracellular amount of IRT or Dox.
Conclusion: This study demonstrates that PEGylated dual-effect liposomes, PL-IRT-Ce6 and PL-Dox-Ce6 offer possible regimens for the treatment of drug-resistant cancer. In the future, the therapeutic effect of the PEGylated dual-effect liposomes will be verified in vivo to examine its efficacy against tumors.
Po-Chun Peng– postdoctoral researcher, Department of Biochemical Science and Technology, National Taiwan University
Chin-Tin Chen– Professor, Department of Biochemical Science and Technology, National Taiwan University