| Summary: | 碩士 === 臺灣大學 === 藥學研究所 === 98 === Background
Photodynamic therapy (PDT) is a novel and minimally invasive treatment method for cancer and non-oncological disorders. PDT is a two-step therapeutic technique in which the topical or systemic delivery of photosensitizing drugs is followed by irradiation with appropriate wavelength. After irradiation with suitable wavelength, then activated photosensitizers transfer energy to molecular oxygen, generating singlet oxygen and reactive oxygen species (ROS) that cytotoxicity species could induce cell death. Since the short diffusion distance (20 nm) and half-life (10-6~10-9 s) of singlet oxygen, direct photodamage commonly occurs at the sites where photosensitizer located. Hypericin (Hyp) is classified as second generation photosensitizer, since it has photosensitizing and fluorescence properties suited for photodynamic therapy and photodynamic diagnosis, in addition, Hyp have high quantum yield of photogeneration of singlet oxygen and relatively low dark toxicity, so Hyp is a potential phototherapeutic agent in photodynamic therapy. However, Hyp forms aggregates in aqueous solution, the hydrophobic nature of Hyp limits its ability to study in vivo and clinical trails.
Objectives
Human hepatoma cell line, Hep3B, was used as in vitro models. We use liposomes and low density lipoprotein (LDL) as Hyp carrier, and study the impacts of different Hyp formulation on cellular uptake, subcellular localization, and Hyp PDT induced cell death mechanisms.
Methods
In this study, Hyp entrapped liposomes composed of 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and cholesterol (Chol) with or without addition of different amounts of cationic lipid ,stearylamine (SA), The follow liposomal formulation were tested：SA0 (molar ratio: 0), SA0.25 (molar ratio: 0.25), SA0.5 (molar ratio: 0.5) and SA1.0 (molar ratio: 1.0).The particle size, Zeta potential and Hyp encapsulation of liposomal Hyp were determined by Zetasizer and spectrofluorometer. The liposomes storage and light irradiation stability were also assessed. In vitro study, we examined cellular uptake capacity of Hyp, Hyp-LDL and liposomal Hyp in serum-free medium for 3h by flow cytometry. We used fluorescence image, MTT assay, flow cytometry and western blotting to observe subcellular localization of Hyp and cell death mechanisms induced by PDT treatment.
Results
Size distribution of liposomes were comparable between four groups for SA0 (183.57 ± 6.80 nm), SA0.25 (181.53 ± 10.55 nm), SA0.5 (174.87 ± 4.26 nm) and SA1.0 (177.90 ± 15.50 nm). In the result of encapsulation efficacy: SA0 (0.094 ± 0.003 μmole Hyp/mg DSPC), SA 0.25 (0.067 ± 0.003), SA 0.5 (0.085 ± 0.006), SA1.0 (0.078 ± 0.010), SA0 and SA0.5 is higher than other groups.
In vitro study, Hep3B incubated with different Hyp formulation for 3h in serum-free medium, the extent of uptake is Hyp＞Hyp-LDL＞SA0＞SA0.25≒SA0.5≒SA1.0. Confocal microscopy confirmed the subcellular localization of different Hyp formulation, Hyp mainly colocalized with endoplasmic reticulum (ER) and Golgi apparatus in all Hyp formulation groups. For Hyp, Hyp-LDL and SA0 groups, the fluorescence was found to be diffuse. However, cationic liposomes show granular fluorescence and some colocalize with lysosome specific dye, LysoSensor. The result of Hyp PDT induced cytotoxicity showed at 24h after Hyp PDT treatment: Hyp＞Hyp-LDL＞SA0＞SA0.25 ≒ SA0.5 ≒ SA1.0, then we used IC50 dose of Hyp (Hyp 28.26 nM、Hyp-LDL 45.26 nM、SA0 83.20 nM、SA0.25 522.89 nM、SA0.5 551.26 nM、SA1.0 635.16 nM) at 2 J/cm2 to do following experiment. After Hyp PDT treatment, compared with control, ctyosolic calcium concentration were significantly increase (Hyp－3.18 ± 0.78 (p＜0.01)、Hyp-LDL－4.33 ± 0.16 (p＜0.001)、SA0－4.29 ± 0.42 (p＜0.001)、SA0.5－3.38 ± 0.50 (p＜0.01)) and the percentage of Sub-G1 phase（apoptosis）of cell cycle were raise (Hyp (22.36 ± 8.67%；p＜0.05)、Hyp-LDL (18.75 ± 7.64%；p＜0.05)、SA0 (16.41 ± 2.97%；p＜0.01)、SA0.5 (18.76 ± 3.42%；p＜0.01) after Hyp PDT treatment, ER-stress marker, C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP-78) were up-regulation, in caspases cascade, caspase-3 and PARP was also cleaved and activated. However, the autophagic marker, LC3I/LC3II (microtubule associated protein light chain 3 I/II), LC3II protein level was not affected by Hyp PDT treatment in Hep3B cell.
Conclusion
In summary, Hyp mainly localized with ER and Golgi apparatus in all Hyp formulation groups, We found Hyp PDT treatment could induce ER-stress (CHOP and GRP-78 were up-regulation) and led to apoptosis (caspase-3 and PARP was also cleaved and activated) in Hep3B cell. Cationic liposomes showed some colocalization with lysosome. However, there is no different between Hyp formulations in apoptosis signal. In the future, in order to improve tissue selectivity and therapeutic efficacy of Hyp formulation, we would study the size, charge and surface modification of liposomes, and the ratio of concentration of LDL to Hyp, there be a great improvement in the application and development in the field of PDT.
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