奈米顆粒熱治療-體外HepG2試驗細胞死亡機制探討及體內試驗小鼠毒性研究

Abstract

近年來磁性奈米顆粒在生醫工程上研究展甚受重視。其應用範圍廣泛，磁性奈米粒子在癌症治療上亦有卓越的貢獻，例如局部注射磁性奈米顆粒至腫瘤區，並外加高週波磁場(AMF)可提高局部腫瘤溫度，利用產生高溫有效控制腫瘤體積，達到癌症的熱治療效果。 本論文的第一部分係探討以磁性奈米顆粒熱治療殺死腫瘤細胞之機制。以共沉澱法合成磁性氫氧基磷灰石奈米顆粒（magnetic hydroxyapatite nanoparticles，mHAPs）做為熱種子，HepG2人類肝癌細胞作為體外試驗對象。磁性氫氧基磷灰石奈米顆粒經X 光與電子束繞射分析為氫氧基磷灰石及四氧化三鐵，SQUID 測得此磁性粒子具有超順磁性。1 mg磁性氫氧基磷灰石奈米顆粒，在高週波磁場條件為 f = 750 KHz, H = 10 Oe 於 1ml 純水中，SLP 值為 181.8 W/g。依據謝勒公式計算XRD數據，氫氧基磷灰石奈米顆粒及四氧化三鐵晶粒大小分別為39.1 nm and 19.5 nm。將HepG2與此磁性粒子共同培養並提供外加磁場，癌症細胞暴露在熱治療溫度(43oC±0.5°C) 30分鐘。WST-1試驗顯示於磁性奈米顆粒熱治療組細胞活性被抑制50%以上，LDH試驗中發現，細胞經過磁性奈米顆粒熱治療處理之後，與控制組差異在三倍以上。在live/dead 染色則呈現與以上試驗相同的結果。HepG2經過磁性奈米顆粒熱治療處理之後，測量到大量的活性氧類 (ROS) 產生。在細胞死亡機制探討的部分，利用Ingenuity pathway analysis (IPA)雲端平台分析cDNA微陣列數據，發現HepG2細胞暴露在此磁性粒子下，DNA損傷相關基因(ATM與GADD45-aplha)被抑制，在訊息傳遞路徑中，p38絲裂原活化蛋白激酶(p38 MAPK)及其上下游 (MKK3/MKK6 及ATF-2)均呈現下調控。這些結果建議，共同以磁性氫氧機磷灰石奈米顆粒與高週波磁場所產生的磁性奈米顆粒熱治療，其產生大量的ROS使HepG2人類肝癌細胞DNA損傷，及熱刺激相關的生物效應導致癌症細胞死亡。

本文第二部分將評估以全身遞送磁性奈米顆粒後磁性熱治療的潛在毒性試驗。以健康之免疫不全裸鼠 (BALB/c Nude mice) 作為試驗對象，將不同濃度的磁性奈米顆粒 (BNF)及磁性奈米顆粒標誌免疫球蛋白G (BNF-IgG) (奈米顆粒濃度分別為1, 3, 5 mg)以尾靜脈注射方式打入小鼠體內，之後將小鼠暴露在不同強度的外加磁場 (400, 600, 800 Oe)。於是奈米顆粒熱處理試驗之後，發現暴露在高劑量的小鼠在胸口上有灼傷的痕跡，小鼠胸口溫度高達51度。進一步確認肝臟外觀也獲得相同的結果。取得肝臟進行蘇木素-伊紅染色，發現暴露在高劑量的小鼠群，肝臟部分區域明顯的壞死，甚至造成死亡。另外肝臟組織以普魯士藍染色也發現，奈米顆粒濃度與肝臟壞死區域有正相關。以血液生化檢查確認肝臟受損情形，發現小鼠暴露在高劑量下，肝臟損傷指標中ALP、AST、LDH及ALT指數均偏高。脾臟則無損傷情形。之後以電感耦合等離子質譜儀法檢測血液、肝臟及脾臟中含鐵濃度。發現沉積在肝臟的鐵濃度，最高達到5000ug 鐵/mg 組織，其數值遠高於脾臟及血液樣品。之後依據組織含鐵濃度計算能量累積，在小鼠暴露於高劑量奈米顆粒熱處理其約1012焦耳。這些結果建議以全身遞送磁性奈米顆粒標誌免疫球蛋白G(BNF-IgG)其所提供奈米熱治療的潛在毒性低於磁性奈米顆粒(BNF)。

Magnetic nanoparticle hyperthermia therapy treatment for cancer has gained more favor in the research community in recent years. However, there are no definitive reports were available on how magnetic nanoparticle hyperthermia induces cancer cell death. In the first part, HepG2 cell death with magnetic nanoparticle hyperthermia (MHT) using hydroxyapatite nanoparticles (mHAPs) and alternating magnetic fields (AMF) was investigated in vitro. The mHAPs were synthesized as thermo-seeds by co-precipitation with the addition of Fe2+. The grain size of HAPs and iron oxide magnetic were 39.1 nm and 19.5 nm were calculated by the Scherrer formula. HepG2 cells were cultured with mHAPs and exposed to an AMF for 30 min yielding maximum temperatures of 43 ± 0.5°C. After heating, cell viability was reduced by 50% relative to controls, lactate dehydrogenase (LDH) concentrations measured in media were three-fold greater than those measured in all control groups. Readouts of toxicity by live/dead staining were consistent with cell viability and LDH assay results. Measured ROS in cells exposed to MHT was two-fold greater than in control groups. Results of cDNA microarray and Western blotting revealed tantalizing evidence of ATM and GADD45 downregulation with possible MKK3/MKK6 and ATF-2 of p38 MAPK inhibition upon exposure to mHAPs and AMF combinations. These results suggest that the combination of mHAPs and AMF can increase intracellular concentrations of reactive oxygen species (ROS) to cause DNA damage, which leads to cell death that complemented heat-stress related biological effects. In the second part, the potential toxicity of magnetic nanoparticle hyperthermia following systemic delivery of magnetic iron oxide nanoparticles (MIONs) was assessed in in vivo study. In this study, 8-week old healthy female nude mice were injected with starch-coated magnetic iron oxide nanoparticles (BNF) or their counterparts labeled with a polyclonal human antibody (BNF-IgG) at 1mg, 3mg or 5mg concentration on day 1. On day 3, animals were exposed to an alternating magnetic field (AMF) having one of three different amplitudes (400, 600 and 800 Oe) for a duration of 20 minutes. 24 hours after AMF treatment, blood, liver and spleen were harvested from each mouse and analyzed with histopathology for tissue damage and with inductively-coupled plasma mass spectrometry (ICPMS) for iron content. Additional endpoints included tissue damage to skin, temperature measurements, and liver function enzyme analysis in serum. Animals treated with different concentrations of BNF/BNF-IgG nanoparticles under different field strength exhibited varying degrees of toxicity. Visible burn lesions were identified on chest region and liver damage after treatment with BNF/BNF-IgG at higher concentration and field strength. Analysis of histopathology revealed wide spread tissue damage when animals were treated with high concentrations of nanoparticles under higher field strength. Following systemic delivery, BNF and BNF-IgG nanoparticles can accumulate in the liver and spleen, making these the sites of potential toxicity. Our findings suggest BNF nanoparticles may be more toxic than BNF-IgG under same alternating magnetic fields.