檸檬酸銨鐵表面改質應用於奈米粒子腫瘤熱治療法

Ferric Ammonium Citrate Nanoparticle Surface Modified for Cancer Hyperthermia

李玉婷(Yu-Ying Li)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10319

摘要

在全球人類死因之中，惡性腫瘤近十五年來都位居第一，在現代社會中，許多因素使癌症細胞滋長，如菸，酒，檳榔，燒烤食物以及工作場所的輻射線…等，甚至壓力過大、煩惱太多等心理因素也會造成癌細胞的產生，且至今仍然沒有方法能夠完全地預防癌症。故醫治技術的開發與改良也成了不可或缺的一項要點。

目前癌症治療的方法常見的有四種：外科手術，放射線治療，化學藥物療法及溫熱療法等，其療法各有其優劣，溫熱療法中又以磁性奈米粒子較能深入針對癌細胞，故本研究主要著重於利用磁性奈米顆粒做為深入生物體的熱治療。

檸檬酸胺鐵(Ferric ammonium citrate)，是一種被FDA認可能適用於人體的材料，由於其順磁性質，有許多研究顯示其材料可以做為MRI的顯影劑，且可大量被人體 (約50 kg) 飲用（用量約1200 mg），而在本研究室過去的研究指出，FAC亦可做為熱治療的磁性奈米粒子，但由於其超親水性容易散失於水性溶劑中(如水)故施打於人體靜脈容易導致其散失於血液中，因此，將檸檬酸胺鐵以明膠做表面改質，並以此作為後續接枝的媒介。

明膠為做奈米材料表面改質常用之原料，其通過FDA認可且有許多優點，如有相對較低的抗體與抗原反應，有許多胺基及醋酸根等官能基，能有效用於表面改質，是一種表面改質常用的材料。

聚乙二醇為一種親水性高分子，表面帶有負電極具有永動性，有助於避免巨噬細胞的吞噬效應及RES反應，因此可增加藥物在血液中存留的時間；葉酸為常見的奈米粒子表面改質之材料，由於肺癌細胞表面有大量的葉酸受體表現，故以葉酸接枝於奈米粒子表面做為標的腫瘤細胞之配合基，有助於增加藥物到達腫瘤細胞的劑量。

本實驗目的為將檸檬酸胺鐵以明膠作為表面改質的媒介並以葉酸及聚乙二醇做奈米粒子表面改質，其實驗方式為利用明膠再交聯過程中所需要用到的戊二醛作為交聯劑結合檸檬酸銨鐵及明膠，並以穿隧式電子顯微鏡、傅立葉紅外線光譜分析、磁滯分析、Ｘ光繞射分析、表面電位及粒徑分析確定材料達到表面改質之目的；以正常細胞 (HFL-1) 及肺癌細胞 (A549) 做LDH及WST-1生物相容性測試，進而以溫熱療法治療及含鐵量分析此兩種細胞。

由磁滯分析、Ｘ光繞射分析及升溫測試可知本材料雖屬順磁性但有良好的升溫效率且在表面改質後與改質前的升溫效率差異不大，由傅立葉紅外線光譜、表面電位及粒徑分析可知，檸檬酸銨鐵藉由明膠做為媒介可與聚乙二醇及葉酸鍵結，達到表面改質之目的。生物相容性實驗的部分，正常細胞 (HFL-1) 及肺癌細胞 (A549) 的LDH及WST-1測試顯示材料幾乎不具有細胞毒性；在細胞與材料共同在外加交流磁場下做42-46 ℃的細胞熱治療可知，肺癌細胞在死亡的數量明顯較正常細胞多，且在細胞與材料共同培養一小時後觀察細胞內含鐵量之結果可知，本材料經由表面改質後堆積於癌症細胞鐵含量也明顯較正常細胞多。故推測是檸檬酸胺鐵經表面改質後進入腫瘤細胞之劑量較正常細胞高，且癌症細胞承受熱治療的能力也明顯比正常細胞差，故檸檬酸銨鐵經由表面改質後可明顯增加其在腫瘤熱治療之效果。

關鍵字

檸檬酸胺鐵 ； 葉酸 ； 明膠

並列摘要

The disease cause major death in human is the malignant tumor. However, the death rate of cancer has been unchanged for the past fifty years. Therefore, cancer therapy has become an important issue in the modern studies.

There are many treatments available for cancer therapy, such as surgery, chemotherapy, radiation therapy and hyperthermia, but these methods have some limitations such as they affect normal cell population as well except hyperthermia. Magnetic nanoparticles induction hyperthermia applies heat to cancers at the deep region in living body and overcomes some disadvantages of conventional cancer hyperthermia.

Because of the magnetic property, ferric ammonium citrate which had been approved by FDA (Food and Drug Adminstration ) and used as contrast agent for MRI (magnetic resonance imaging). Previous studies in our lab describe that ferric ammonium citrate can be also used as a magnetic particle for hyperthermia but it is very easily hydrolyzed. Therefore, it was concluded that surface immobilization of ferric ammonium citrate by gelatin may prolong the period of FAC in the blood circulation.

However, the surface modified nanoparticle for intravenous injection has to overcome RES and macrophage clearance in order to prolong the period in blood circulation and target to specific site.

The purpose of this study is to immobilize ferric ammonium citrate by gelatin and to surface modified by PEG-FA. In this study, gelatin immobilized ammonium ferric citrate was prepared by glutaraldehyde as cross-linking agent. PEG and FA used for material surface modification ware prepared by EDAC and NHS to crosslink PEG-FA and for AFC-Gel surface modification. The surface modification was characterized by TEM, FTIR, SQUID, XRD and zeta potential analysis. In addition, LDH and WST-1 are used to analysis the cytotoxicity of HFL-1 and A549. The uptake and hyperthermia in vitro test are used the analysis the efficiency of surface modified material target to the cells.

From the SQUID and XRD result, the FAC shows paramagnetic property. From TEM, FTIR, and Zeta potential analysis, the material has been crosslinked with surface modified by PEG and FA. From the LDH and WST-1 results, both FAC and surface modified FAC are not showed any significant difference on HFL-1 and A549. However, when surface modified FAC co-culture with HFL-1 and A549 under external alternative field for hyperthermia (42-46 ℃) showed significant cytotoxic on A549 but not on HFL-1. FAC with and without surface modification co-culture with HFL-1 and A549 and analysis the ferric ion by Prussian blue showed significant difference on A549 with surface modified FAC but not on FAC without surface modification. This result shows that FAC with surface modification will tend to retain lung cancer cells (A549) more than in normal cells (HFL-1) that cause lung cancer cells with surface modified FAC under external alternative field showed significant toxicity. In addition, lung cancer cells have lower heat resistance ability, decrease more than the normal cell under hyperthermia treatment.

並列關鍵字

Hyperthermia ； magnetic ； ammonium ferric citrate ； folic acid

參考文獻

4. László Tabár MDa, B.V.M., Hsiu-Hsi Chen PhDd, Stephen W. Duffy MSce, Ming-Fang Yen MScd, Ching-Feng Chiang BScd, Ulla Brith Krusemo BScf, Tibor Tot MDb and Robert A. Smith PhDg, THE SWEDISH TWO-COUNTY TRIAL TWENTY YEARS LATER: Updated Mortality Results and New Insights from Long-Term Follow-up Radiologic Clinics of North America, 1 July 2000. 38(4): p. 625-651

5. J. Ewing, W.B.S., Philadelphia, , Neoplastic Diseases: A Treaties on Tumors 4th ed. 1940.

7. C. J. Marshall, E.A.N., Oncogenes and cell proliferation: Cancer genes: lessons from genetics and biochemistry Curr Opin Genent Dev, 1998. 8(1): p. 11-3.

9. Connell, J.E. and D.C. D, Risk of ciguatera fish poisoning: impact on recommendations to eat more fish. Asia Pac J Clin Nutr, 2003. 12 Suppl: p. S67.

11. Henschke CI, Y.D., Libby DM, Pasmantier MW, Smith JP, Miettinen OS, Surivival of patients with stage I lung cancer detected on CT screenung. N Engl J Med 2006, 2006. 355: p. 1763-1771.