| 摘要: | 背景: 隨著癌症發生人數逐年增加，因此癌症的診斷和治療亦逐年受到重視。光動力療法(Photodynamic therapy, PDT)是眾多治療癌症的技術之一。光動力療法是一種非侵入性的治療癌症的方法。然而，實體腫瘤缺氧部位的診斷和提高光的組織穿透能力是目前必須克服的挑戰。
研究目的: 結合近紅外(Near infrared, NIR)激發的光敏劑和具有大斯托克位移(Stokes shift)的硝基還原?探針，並以線粒體為靶點，可用於深入的組織體外評估，測量低氧條件下的水平與腫瘤定位。本研究將從巨觀和微觀角度測試和分析自製光敏劑對細胞性能的PDT效果，包括細胞存活率、增殖能力，以及活性氧(Reactive oxygen species, ROS)水平。此外，還將調查具有大斯托克位移的硝基還原?探針的細胞內共定位。我們假設自製的硝基還原?探針能夠區分常氧和缺氧條件，並且使用針對線粒體的光敏劑進行PDT可能會導致細胞凋亡。
材料與方法: 硝基還原?探針透過UV-vis/NIR分光光度計和螢光分光光度計進行鑑定，分別確定哪種溶劑可以適當地將其溶解並表達最佳激發波長。在體外測試中，使用具有較高或較低放大倍率的倒立式螢光顯微鏡可以在巨觀或單一細胞層級的視野下來識別來自硝基還原?探針、光敏劑或專一性染劑(例如羅丹明123、hoechst 或mitotracker)的螢光訊號。PDT對細胞的表現測試中，會利用波長830 nm的雷射照射細胞，並測試添加光敏劑前後對細胞表現的影響，其中包含細胞活性、增殖(Proliferation)能力、活性氧物質(Reactive oxygen species)的生成，以及肌動蛋白絲(Actin filament)表現的測試。
結果與討論: 硝基還原?探針溶於甲醇時具有最高的吸光度，其吸光密度(Optical density, OD)值為0.58 L/(g·cm)，而在溶解在H2O時的吸光度最低，其OD值為 0.40 L/(g·cm)。在螢光測試中，無論溶解在乙醇或水中，以400 nm波長激發時均表現出最高強度。體外細胞毒性試驗中，光敏劑的IC50為16.25 μM，硝基還原?探針的IC50則為74.28 μM。在厭氧誘導後，細胞有產生缺氧誘導因子-α (Hypoxia-inducible factors-α)，且細胞在缺氧狀態下活力均會降低。在偵測細胞厭氧狀態之前，本研究觀察細胞厭氧前後的表現變化。A549細胞在缺氧誘導後，其粒線體碎片計數(Mitochondria fragmentation count, MFC)以及粒線體膜電位訊號雜訊比(Signal-to-noise ratio, SNR)皆有增加的趨勢。此外，硝基還原?探針在缺氧誘導後的A549細胞中可顯示顯著的螢光訊號。在共軛焦雷射顯微鏡結果中發現，以波長458 nm激發硝基還原?探針可在605 nm到650 nm波段範圍間收到螢光訊號，其結果表示出其有斯托克斯位移(Stokes shift)高達150 nm。此外，在共定位(colocalization)的測試中的皮爾森積差相關係數(Pearson correlation coefficient)共定位分析為0.988。隨後，使用放大倍率更高的90倍放大倍率的螢光倒置顯微鏡更詳細地捕捉到單細胞內部結構，其皮爾森相關係數降到0.442。與巨觀的分析結果相比，單一細胞層級的觀察可以透露出較多的結構與信號的細微觀察。在PDT的效果測試中，已證明在不添加光敏劑的情況下，使用不同劑量的830 nm近紅外線雷射會導致不同的細胞活力表現。而當劑量為10 J/cm2的時候，其細胞活性與控制組已無顯著差異，為日後作為添加光敏劑做光動力治療時的參照照光劑量。而若添加光敏劑之後，當照光劑量達到15 J/cm2，無論是在厭氧或是常氧的狀態下，細胞活性皆有顯著性地下降。關於ROS的變化量，當照光劑量會增至15 J/cm2時，ROS水平增加。在單細胞層面上，細胞形態和肌動蛋白絲的變化顯示細胞收縮，且在30分鐘內ROS產量增加。因此，使用單細胞層級分析方法有助於說明細胞在PDT治療之後的微妙變化。
結論: 與常氧情況相比，A549細胞在缺氧情況下會表現出較低的細胞活力並引起粒線體功能的改變。硝基還原?探針可以顯示出高斯托克斯位移，與粒線體的高度重和，以及在濃度20 μM時可顯示出更強的螢光訊號來偵測缺氧情況。在厭氧與常氧的條件下粒線體的結構和訊號變化時發現粒線體的型態與訊號皆會發生變化。當利用細胞暴露於不同劑量的NIR雷射中會使細胞產生不一樣的表現。光照劑量為15 J/cm2的對癌細胞具有較佳的光動力治療效果，因為具有良好的抑制細胞活力和增加ROS產生的功效，以抑制癌細胞。本研究中，利用單細胞技術應用在硝基還原?的偵測或是光動力治療的功效評估實驗可放大細節處，並解釋巨觀觀察中難以觀察到的現象。
Background: As the increase population for getting the cancer, the diagnosis and therapy for cancer are urgently needed. Photodynamic therapy (PDT) is one of the technology to treat the cancer, which is an non-invasive way to treat the cancer. However, the diagnosis in hypoxia site in solid tumor and the lower efficiency of deeper penetrating are the issues that have to be overcome.
Purpose of study: Combining the near infrared (NIR) triggered photosensitizer and a nitroreductase probe with large Stokes shift targeting on the mitochondria could be developed for treating with a deeper tissue in vitro assessment and measuring the level in hypoxia condition, localizing the tumor. This study will use both macroscopic and microscopic perspectives to test and analyze the photodynamic therapy (PDT) effects of a homemade photosensitizer on cell performance, including cell viability, proliferation, and reactive oxygen species (ROS) levels. Additionally, it will investigate the intracellular colocalization of a nitroreductase probe with a large Stokes shift.We hypothesize that the homemade nitroreductase probe can distinguish between normoxic and hypoxic conditions, and that the PDT effect using a mitochondria-targeted photosensitizer may lead to cell apoptosis.
Materials and methods: The nitroreductase probe would be identified by UV-Visible/NIR spectrophotometer and spectrofluorometer to identify which kind of the solvent could dissolve it appropriately and the best excitation wavelength, respectively. The in vitro test utilized inverted fluorescence microscopy or confocal microscopy with different observation levels to identify fluorescence signals from the nitroreductase probe, photosensitizer, and the dyes for staining such as rhodamine 123, hoechst 33342, or mitotracker red, which would be used in both bulk and single-cell levels. For evaluating the PDT effect before and after adding the photosensitizer, cell viability, proliferation ability, reactive oxygen species level, and actin performance would be tested in bulk or at single-cell level, which would be triggered with 830 nm NIR laser.
Result and discussion: The highest absorbance for nitroreductase probe while dissolving in methanol is 0.58 L/(g·cm). The lowest absorbance for nitroreductase probe while dissolving in H2O is 0.40 L/(g·cm). In the fluorescence test, it displayed the highest intensity while using 400 nm wavelength for excitation, regardless of whether it was dissolved in ethanol or H2O. In vitro, cell toxicity tests showed that the IC50 for photosensitizer is 16.25 μM, while the IC50 for nitroreductase probe is 74.28 μM. The cell viability would decrease while suffering the hypoxia condition, and it showed the hypoxia-inducible factors-α (HIF-α) secretion after hypoxia induction. Before detecting the hypoxia level in cells, testing the cellular behavior change before and after hypoxia. After cells suffered in hypoxia induction, both signal-to-noise ratio (SNR) of the membrane potential and the mitochondrial fragmentation count (MFC) increase. The nitroreductase probe successfully displayed a fluorescence signal under hypoxic conditions. Confocal microscopy demonstrated that the probe could emit fluorescence in the range of 605 to 650 nm when excited with a 458 nm laser after hypoxia induction, indicating a Stokes shift of approximately 150 nm. In addition, the Pearson correlation coefficient, which could evaluate the colocalization with mitochondria, is 0.988. Afterward, fluorescence inverted microscopy with 90X magnification with the higher resolution was used to capture the single-cell inner structure in more detail, and the Pearson correlation coefficient came to 0.442. Compared to bulk level analysis, single-cell level provides more detailed insights into both structure and signal. Regarding the PDT effect, it showed that varying doses of an 830 nm NIR laser affect cell viability even without a photosensitizer. At 10 J/cm?, there was no significant change in cell viability, which was used in PDT experiments with the photosensitizer. However, when the photosensitizer was added, cell viability significantly decreased compared to the control group under both normoxic and hypoxic conditions at a dose of 15 J/cm?. Regarding ROS level changes, ROS levels increase when the dosage reaches 15 J/cm?. At the single-cell level, changes in cell morphology and actin filaments indicate cell shrinkage and increased ROS production within 30 minutes. Single-cell analysis effectively reveals these subtle changes after PDT.
Conclusion: Compared to the normoxia situation, A549 would show lower cell viability and cause a change in mitochondrial function under the hypoxia situation. Nitroreductase probe could also show the significant Stokes shift, good correlation with mitochondria and a stronger fluorescence signal to detect the hypoxia situation with a concentration of 20 μM. Using different dosages of the NIR laser to treat the cells would cause different cell performances, and it is better to use 15 J/cm2 to treat the cancer cells for PDT because of the good efficacy for inhibiting cell viability and increasing ROS production to approach the cancer killed. In both nitroreductase detection and PDT efficacy evaluation experiments, single-cell approaches revealed details that might not be explained clearly at the bulk level. |
