脫鎂葉綠酸接枝奈米金粒子在人類皮膚癌細胞光動力治療之應用

Pheophorbide-conjugated Gold Nanoparticles for Photodynamic Therapy of Human Epidermoid Carcinoma A431 Cells

陳政保(Cheng-Pao Chen)

指導教授 ： 李文婷

中原大學/理學院/奈米科技碩士學位學程/碩士(2012年)

https://doi.org/10.6840/cycu201200649

摘要

本研究目的在以水相合成的奈米金粒子 (Gold nanoparticles, GNPs) 作為脫鎂葉綠酸 (Pheophorbide a, Pho a) 的遞送載體，並探討其在人類皮膚癌 A431 細胞之光動力治療 (Photodynamic therapy, PDT) 應用。首先製備出Large-size GNPs (l-GNPs)、Medium-size GNPs (m-GNPs) 及Small-size GNPs (s-GNPs) 三種粒徑的 GNPs。分析GNPs 對 A431 細胞之生物相容性，結果發現不同濃度l-GNPs、m-GNPs及 s-GNPs 在與 A431 細胞共培養 24 小時後，細胞存活率皆在 97 % 以上；l-GNPs 及 m-GNPs的乳酸脫氫酶 (Lactate dehydrogenase, LDH) 釋放量在 10 % 以內。然而 3.0 μg/mL s-GNPs 會造成A431 細胞 LDH 釋放率增加至 17 %，而此濃度之 s-GNPs 並不會使細胞內活性氧分子 (ROS) 含量增加。利用軟性瓊脂生長測試觀察 GNPs 對細胞長期增生之影響，發現三種 GNPs 皆會使細胞群落數目降低。接著合成 s-GNPs-Pho a 與 m-GNPs-Pho a，並以紫外光/可見光光譜、傅立葉轉換紅外線光譜及螢光光譜分析，確認已成功將 Pho a 接枝於 GNPs 上。以波長 660 nm、能量 10.2 J/cm2 對 A431 細胞進行 PDT，發現 s-GNPs-Pho a與m-GNPs-Pho a的半致死劑量 (LD50) 分別為 6.09±1.37 ng/mL及 7.18±0.88 ng/mL；而Pho a 之 LD50則為4.00±0.71 ng/mL。當藥物濃度在 50 ng/mL 時，三者之細胞死亡率皆高達 95%；此外，在藥物濃度 50 ng/mL，Pho a 的 ROS 產量為控制組的 3 倍，s-GNPs-Pho a與 m-GNPs-Pho a 皆為控制組的 1.2 倍。觀察細胞對藥物的攝取量，發現 Pho a 在 24 小時可達細胞攝取之最大量，s-GNPs-Pho a與m-GNPs-Pho a 的細胞最高攝取量分別在 4 小時與 12 小時。綜上所述，Pho a 與 GNPs會改變 Pho a 進入細胞的方式，而接枝於 GNPs 上的Pho a在細胞內的累積量則受到GNPs 直徑的影響，進而會影響其在 PDT 應用的效率。

關鍵字

人類皮膚癌細胞 ； 光動力治療 ； 奈米金粒子 ； 光感藥物 ； 脫鎂葉綠酸 a

並列摘要

The objectives of the study were to investigate the potential application of water-soluble gold nanoparticles (GNPs) as pheophorbide a (Pho a) delivering vectors for photodynamic therapy (PDT) against human epidermoid carcinoma A431 cells. Large-size GNPs (l-GNPs), medium-size GNPs (m-GNPs) and small-size GNPs (s-GNPs) were first synthesized. Biocompatibilities of GNPs at different concentrations were evaluated. The results found that the viabilities were higher than 97% when A431 cells were incubated with s-GNPs, m-GNPs and l-GNPs, whereas the release rates of lactate dehydrogenase (LDH) were less than 10% with m-GNPs and l-GNPs. When A431 cells were incubated with 3.0 μg/mL s-GNPs, LDH release increased up to 17% in which no apparent increase of reactive oxygen species (ROS) was observed. Soft agar colony formation assay was used to determine the long-term effect of GNPs on A431 cells proliferation. As a result, the numbers of colony formation decreased as the concentration increased with three sizes of GNPs. Next, the successful conjugation of Pho a onto the surface of GNPs to produce s-GNPs-Pho a and m-GNPs-Pho a was confirmed by ultraviolet/visible spectrophotometric analysis, Fourier transform infrared spectroscopic analysis and fluorescence spectroscopic analysis. Half lethal dosage (LD50) of unconjugated Pho a, s-GNPs-Pho a and m-GNPs-Pho a were 4.00±0.71 ng/mL, 6.09±1.37 ng/mL and 7.18±0.88 ng/mL, respectively, when combined with 10.2 J/cm2 and 660 nm PDT. PDT cytotoxicity of Pho a, s-GNPs-Pho a and m-GNPs-Pho a at 50 ng/mL were 95%. In addition, PDT with Pho a, s-GNPs-Pho a and m-GNPs-Pho a at 50 ng/mL resulted in 3.0, 1.2 and 1.2 fold increase of ROS production, respectively, compared to that of the control group. The cellular uptake study showed that the maximal accumulation of unconjugated Pho a, s-GNPs-Pho a and m-GNPs-Pho a were at 24, 4 and 12 hours, respectively. In conclusion, Pho a and GNPs conjugation changed the way of Pho a into A431 cells. The amount of GNPs-Pho a accumulated inside the cells depended on the diameter of GNPs, thereby influenced the efficacy of PDT.

並列關鍵字

human epidermoid carcinoma cell ； photodynamic therapy ； gold nanoparticle ； photosensitizer ； pheophorbide a

參考文獻

[75] 范瑞鴻 (2008) 奈米金粒子對於人類細胞之生物相容性探討。中原大學碩士學位論文.

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