結合共沈澱及預凝膠法合成氧化鐵/海藻膠奈米粒子 並用於肝癌細胞的標靶溫熱治療

Synthesis of Iron(II, III) Oxide/Alginate Nanoparticles through a Combined Pre-gel and Co-precipitation Method for Targeted Liver Cancer Hyperthermia

廖士翔(Shih-Hsiang Liao)

指導教授 ： 吳嘉文

國立臺灣大學/工學院/化學工程學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00457

摘要

本研究的目的是合成親水性、分散性良好且能標靶人類肝癌細胞HepG2 (Human hepatocellular carcinoma cell line)的磁性奈米粒子，並用外加交流磁場進行溫熱治療，以達到殺死癌細胞的效果。結合預凝膠(Pre-gel)與共沉澱法(Co-precipitation)，能藉由調整pH值與海藻膠量合成出流體力學直徑(Hydrodynamic diameter)為122 nm到560 nm的Fe3O4@Alg奈米複合材料，此合成方法無需多步驟合成且皆在水溶液中反應，用於生物體內較為安全。由於海藻膠上富含羥基(−OH)與羧酸基(−COOH)，提供材料表面高負電性與親水性，因此材料合成好即可在水中長期穩定分散，且羧酸基能進一步修飾上標靶分子。海藻膠包覆的磁性奈米粒子經由EDC/NHS活化粒子表面的羧酸基，使其與標靶分子(Folic acid和D-galactosamine)上的一級胺基產生胜肽鍵，使磁性奈米粒子成為擁有folic acid和D-galactose兩種標靶分子的Fe3O4@Alg-FA和Fe3O4@Alg-(D-Gal)，以標靶人類肝癌細胞HepG2。
藉由測量到表面電位的下降、羧酸基的C-O伸縮震動峰的消失與偏移和高溫(800°C)處理後重量百分比的改變來確認材料中有海藻膠與標靶分子，並推算各成分的重量百分比。經SQUID測量材料磁性近似超順磁性(Superparamagnetism)且飽和磁化強度皆約在70 emu/g。接有標靶分子的材料(濃度0.5 mg/mL)經高週波產生的磁場(780 kHz；99 kA/m)加熱純水，測得specific absorption rate (SAR)皆為308.4 W/gFe。利用ICP-MS量測發現接上folic acid後材料在細胞中的Fe3O4累積量多了15.4倍，而接上D-Galactose後多了19.7倍。材料藉由高週波產生的磁場在細胞內加熱進行溫熱治療，Fe3O4@Alg-FA和Fe3O4@Alg-(D-Gal)分別能使細胞的相對存活率降為0.1和0.04，比起Fe3O4@Alg的0.61有更好的療效，證實接有標靶分子的Fe3O4@Alg能有效的累積在細胞內，使溫熱治療殺死HepG2細胞的效果更好。最後測量材料的血漿凝血時間(Plasma clotting time)與進行溶血試驗(Hemolytic test)，證實材料不易造成血液凝血和溶血，能安全用於生物體內。

關鍵字

溫熱治療 ； 葉酸 ； 半乳糖 ； 預凝膠 ； 海藻膠 ； 四氧化三鐵

並列摘要

The purpose of this study is to synthesize well-dispersed and hydrophilic magnetic nanoparticles that can target HepG2 (Human hepatocellular carcinoma cell line). Hyperthermia to kill cancer cells can be achieved by applying an AC magnetic field. A combined pre-gel and co-precipitation method can adjust the hydrodynamic diameter of Fe3O4@Alg nanoparticles from 552 nm to 122 nm by varying the pH value and amount of sodium alginate. This method does not need a multi-step synthesis and synthesis in an aqueous solution is more suitable for in vivo usage. Since alginates are rich in hydroxyl (−OH) and carboxylic acid (−COOH) groups, they can provide a highly negative surface charge and make the particle hydrophilic. Alginate can stabilize the dispersion of Fe3O4 in water, and the carboxylic acid groups can be further modified with targeting molecules. Alginate coated magnetic nanoparticles further modified by EDC/NHS activated carboxylic acids will react with the primary amine groups of the targeting molecule (Folic acid and D-galactosamine) to become amino peptide bonds. The magnetic nanoparticles then become Fe3O4@Alg-FA and Fe3O4@Alg-(D-Gal), so these nanoparticles can target HepG2 cell.
Through a decrease in the zeta potential, as well as a disappearance and multiple shifts in the C−O stretching vibration peaks, we can confirm that alginate and targeting molecules are bonded on the material. Calculation of the wt% of each component can be done using the remaining wt% after heating to 800°C. With SQUID measurements, the materials almost have superparamagnetism properties, and the saturation magnetization is about 70 emu/g. The magnetic field generated by the high frequency (780 kHz; 99 kA/m) causes the targeting-functionalized materials (concentration 0.5 mg/mL) to heat water, and the specific absorption rate (SAR) was detected to be 308.4 W/gFe. Using ICP-MS, we found that the accumulation of Fe3O4 in HepG2 for Fe3O4@Alg-FA and Fe3O4@Alg-(D-Gal) are 15.4 and 19.7 times respectively more than that of Fe3O4@Alg. The materials generate heat in HepG2 through an applied magnetic field, leading to hyperthermia. The relative cell viabilities of the Fe3O4@Alg-FA and Fe3O4@Alg-(D-Gal) groups are 0.1 and 0.04, respectively, which is better compared to Fe3O4@Alg (0.61). This result confirms that targeting molecules help Fe3O4@Alg accumulate in cells effectively, resulting in an increased chance for the death of HepG2. Lastly, measurements of the plasma clotting time of the material and hemolytic tests confirmed that the material does not speed up blood clotting and trigger hemolysis, which means that it is safe to be utilized in vivo.

並列關鍵字

hyperthermia ； folic acid ； D-galactose ； pre-gel ； alginate ； iron(II, III) oxide

參考文獻

[80] 梁永和. 以預凝膠法製備磷酸鈣/海藻膠之核/殼奈米粒子並應用於酸鹼應答型的藥物釋放: 國立台灣大學碩士論文; 2012.

[1] Meade VM, Burton MA, Gray BN, Self GW. Distribution of different sized microspheres in experimental hepatic tumours. European journal of cancer & clinical oncology. 1987;23:37-41.

[2] Yamada R, Kishi K, Sato M, Sonomura T, Nishida N, Tanaka K, et al. Transcatheter arterial chemoembolization (TACE) in the treatment of unresectable liver cancer. World J surg. 1995;19:795-800.

[3] Kettenbach J, Stadler A, Katzler I, Schernthaner R, Blum M, Lammer J, et al. Drug-loaded microspheres for the treatment of liver cancer: Review of current results. cardiovasc intervent radiol. 2008;31:468-76.

[4] Moroz P, Jones SK, Gray BN. Status of hyperthermia in the treatment of advanced liver cancer. Journal of surgical oncology. 2001;77:259-69.