金絲桃素微脂粒製劑於光動力治療之細胞死亡機制探討

A Study on the Cell Death Mechanisms of Liposomal Hypericin in Photodynamic Therapy

溫裕瀚(Yu-Han Wen)

指導教授 ： 何藴芳

國立臺灣大學/醫學院/藥學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00685

摘要

研究背景
光動力治療（photodynamic therapy；PDT）是一種新興且低侵入性的治療方法，可應用於癌症及其它病症的治療。光動力治療可分為兩個步驟，含光敏感劑經由局部或全身性給藥並給予適當波長的光照。光敏感劑在適當波長的激發後，便會透過能量傳遞產生單態氧及活性氧分子，藉由這些毒性分子的產生，可導致細胞的死亡。但因單態氧的擴散距離短（約20 nm）且持續時間短（約10-6~10-9 s），所以光敏感劑於胞內的分布會決定光照後首要傷害的部位。金絲桃素（hypericin；Hyp）屬於第二代光敏感劑，具有光敏感與螢光的特性，適用於光動力治療及光動力診斷（photodynamic diagnosis），此外，Hyp具單態氧高產率及低暗毒性等優點，故Hyp被認為在光動力治療應用上深具潛力；但因Hyp高脂溶性，在水溶液中易產生聚集（aggregate）的現象，將不利Hyp應用於未來動物或臨床試驗的進行與發展。

研究目的
本研究中，以人類肝癌細胞Hep3B做為體外研究模式的對象。我們利用微脂粒及低密度脂蛋白（low density lipoprotein；LDL）做為Hyp的藥物載體，並且研究各類Hyp製劑在細胞攝取、胞內分布及光動力處理後所引起細胞死亡機制的差異。

研究方法
本研究中以1,2-distearoyl-sn-glycero-3-phosphocholine（DSPC）及膽固醇做為微脂粒的主要材料，並添加不同比例的正電荷脂質stearylamine（SA），依SA所佔之莫耳比分為SA0（莫耳比0）、SA0.25（莫耳比0.25）、SA0.5（莫耳比0.5）及SA1.0（莫耳比1.0）等四類liposomal Hyp。製備之liposomal Hyp再以粒徑分析儀及螢光光譜儀分別進行粒徑及包覆率的測定。在微脂粒粒徑穩定性之分析，分別以保存穩定性及光照穩定性檢定，以供確保後續細胞實驗進行期間，微脂粒的粒徑圍完整且均一的。
在細胞實驗中，以流式細胞儀分析Hep3B細胞與各類Hyp製劑於serum-free條件下處理3小時的攝取情形，我們利用螢光影像分析、MTT細胞毒性實驗、流式細胞儀及西方墨點法，觀察各類Hyp製劑於胞內的分布及後續光照處理後引起細胞死亡機制的探討。

研究結果
SA0、SA0.25、SA0.5及SA1.0等各類微脂粒制劑之粒徑依次為183.57 ± 6.80 nm、181.53 ± 10.55 nm、174.87 ± 4.26 nm、177.90 ± 15.50 nm。Hyp包覆率分別為0.094 ± 0.003 μmole Hyp/mg DSPC、0.067 ± 0.003、0.085 ± 0.006及0.078 ± 0.010，其中以SA0及SA0.5的包覆率較高。
在Hyp細胞攝取之研究中，Hep3B細胞在serum-free培養液條件下六類Hyp製劑培養3小時，Hyp攝取量之大小比較為：Hyp＞Hyp + 2.5 μg LDL/mL（Hyp-LDL）＞SA0＞SA0.25 ≒ SA0.5 ≒ SA1.0。在Hep3B和各組Hyp製劑處理3小時後，發現各組製劑均主要以分布在內質網與高基氏體為主，但相較於正電荷liposomal Hyp（SA0.25、SA0.5、SA1.0）等三類製劑組，Hyp組、Hyp + 2.5 μg LDL/mL組及liposomal Hyp（SA0）組中，Hyp之胞內分布較均勻散布於細胞質；而三種正電荷微脂粒組中，Hyp之胞內分布則呈現顆粒狀，且有部份和溶酶體染劑（LysoSensor）產生colocalization的現象。在光動力處理後24小時，發現各組Hyp製劑之細胞毒性大小依序為：Hyp＞Hyp-LDL＞SA0＞SA0.25 ≒SA0.5 ≒ SA1.0，在2 J/cm2光照下，採用各組Hyp的 IC50進行後續的實驗，分別為：Hyp組 28.26 nM、Hyp-LDL組 45.26 nM、SA0組 83.20 nM、SA0.25組 522.89 nM、SA0.5組 551.26 nM、SA1.0組 635.16 nM。各正電荷微脂粒組在細胞攝取、胞內分布及細胞毒性的實驗上，彼此間無顯著差異，故僅以Hyp包覆濃度及包覆率較高的SA0.5製劑進行細胞凋亡機制探討。在Hyp組、Hyp-LDL、SA0及SA0.5各組光動力處理後30分鐘即可發現Hep3B細胞內鈣離子濃度均有明顯上升，各組Hyp製劑和控制組相比均有明顯差異：3.18 ± 0.78（p＜0.01）、4.33 ± 0.16（p＜0.001）、4.29 ± 0.42（p＜0.001）、3.38 ± 0.50（p＜0.01）。在光照處理後24小時，Hep3B細胞週期並無停滯現象，但四組之Sub-G1期（細胞凋亡）和控制組相比均有明顯增加（Hyp組（22.36 ± 8.67%；p＜0.05）、Hyp-LDL組（18.75± 7.64%；p＜0.05）、SA0組（16.41 ± 2.97%；p＜0.01）、SA0.5（18.76 ± 3.42%；p＜0.01））。為了進一步探究四種Hyp製劑光動力處理後之細胞死亡機制，發現各組均可見endoplasmic reticulum (ER)-stress指標蛋白C/EBP homologous protein（CHOP）及glucose-regulated protein 78（GRP-78）的活化，在細胞凋亡相關蛋白可見到caspase-3及PARP被切割且活化的現象，顯示Hyp光動力處理後將會透過ER-stress引起Hep3B細胞產生細胞凋亡。在細胞自噬指標蛋白LC3I/LC3II（microtubule associated protein light chain 3 I / II）蛋白表現上，並未發現在Hyp光動力處理後LC3II表現有增加的現象，故在本研究處理條件下，Hyp光照處理並未發現Hep3B細胞自噬的現象。

結論
在本研究中發現，各種Hyp製劑於胞內分布主要傾向分布於內質網及高基氏體，各類Hyp製劑光動力處理後將會透過ER-stress（CHOP及GRP-78的活化）引起Hep3B細胞產生細胞凋亡（caspase-3及PARP被切割且活化）。此外，正電荷微脂粒有部份和溶酶體產生colocalization的現象，光動力處理後可能會導致後續細胞死亡機制的不同，但在後續細胞死亡機制的探討上，與其他類製劑組間並無明顯差異。未來如能就Hyp製劑的組織與細胞選擇性及光動力治療效率深入探討，在微脂粒的粒徑、電荷、表面修飾及低密度脂蛋白和Hyp間比例的設計最佳組合，將期能夠在PDT的開發及應用上有所進展。

關鍵字

光動力治療 ； 金絲桃素 ； 低密度脂蛋白 ； 微脂粒 ； 細胞內分佈 ； 細胞凋亡

並列摘要

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.

並列關鍵字

photodynamic therapy ； hypericin ； low density lipoprotein ； liposomes ； subcellular localization ； apoptosis.

參考文獻

65. 楊穎奇, 金絲桃素於人類肝癌細胞的攝取路徑與細胞內分佈之探討, in 藥學研究所, 臺灣大學. p. 104.

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