評估奈米氧化鐵-氫氧基磷灰石-順鉑藥物載體應用於肺癌之熱治療與化療之合併療法之研究

The characterization and evaluation of cisplatin-loaded magnetite-hydroxyapatite nanoparticles as dual treatment of hyperthermia and chemotherapy for lung cancer therapy

黃建元(Jian-Yuan Huang)

指導教授 ： 林峰輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.02671

摘要

近三十年來惡性腫瘤一直是國內十大死因排名之首，其中肺癌更居所有癌症之冠。儘管當今之醫療水準已有相當的進展，肺腺癌的平均五年之存活率依舊難以有效率的增加。化療一直是治療肺癌的標準療程之一，其中第一線化療用藥順鉑(cisplatin, CDDP)或其混合處方對非小細胞肺癌有不錯的療效。由於順鉑在使用上常有抗藥性的問題，所以需合併其他治療方式提高療效以及克服抗藥性。
本研究為利用氧化鐵-奈米氫氧基磷灰石-順鉑磁性藥物載體於肺癌合併化療與熱治療之研究，達到協同增效(synergistic effect)的治療作用。奈米氫氧基磷灰石(HAp)具有良好的生物相容性與生物可分解性，其已廣泛應用於生物醫學領域。由於傳統的共沉法無法有效地將藥物載於氫氧基磷灰石上，故本實驗利用微乳化法來合成氫氧基磷灰石-順鉑奈米粒子(HAp/CDDP)能有效將達到22.9%順鉑載藥率。再利用鹼性環境下將氧化鐵共沉在氫氧基磷灰石-順鉑奈米粒子上形成氧化鐵-氫氧基磷灰石-順鉑奈米粒子(mHAp/CDDP)。此磁性粒子晶體主結構經由X光與電子束繞射分析為氫氧基磷灰石。氧化鐵的結構為磁鐵礦。經由穿透式電子顯微鏡可得知所合成之奈米晶格大小範圍介於50 – 80 nm。由SQUID測得mHAp/CDDP為超順磁性。在37ºC以及磁場條件為f = 750 KHz, H = 10 Oe時，SAR值為72.91W/g。此磁性粒子在磁場的作用下具有足夠的能力使溫度由室溫在10分鐘上升20ºC。
經由穿透式電子顯微鏡可得知磁性粒子與人類肺腺癌細胞(A549)共培養，細胞會藉由吞噬作用將奈米粒子內吞至核內體(endosome)內。共培養12小時候，以共軛顯微鏡觀察，奈米粒子在與細胞會分布在溶小體(lysosome)內。由於溶小體內為酸性環境會將氫氧基磷灰石奈米粒子溶解並產生滲透壓將溶小體撐破釋出化療藥物。因此順鉑將能進入到細胞核內與DNA結合形成一複合體影響細胞正常複製生長，進而造成癌細胞走向凋亡。
由細胞相容性實驗可發現，HAp與mHAp具有良好的生物相容性，但HAp/CDDP與mHAp/CDDP會持續釋出順鉑造成細胞死亡。再搭配熱治療實驗能發現熱療與化療能造成之協同增效作用提高抗癌效果，實驗三天後儘13%癌細胞存活，療效明顯比單一治療方法高。由西方點墨實驗(Western blot)得知，在研究中所釋出的順鉑會透過胞外訊號調節激酶(Extracellular signal-regulated kinases, ERK)途徑造成細胞凋亡(apoptosis)。
在小鼠動物實驗中，以免疫不全裸鼠(BALB/c Nude mice)後肢皮下植入A549細胞作為癌症動物模式。將小鼠分組成六組不同之治療方式，其中施打mHAp/CDDP並暴露於外加磁場下的組別其療效最佳，腫瘤生長明顯受到抑制。此外，本研究方式並未對實驗動物造成肝腎毒性以及其他的副作用。因此本研究所開發之氧化鐵-氫氧基磷灰石-順鉑奈米粒子是一種同時包含化療與熱療於單一奈米系統上，其具有增效作用應用於抗癌研究上。

關鍵字

肺癌治療 ； 熱治療 ； 氫氧基磷灰石 ； 微乳化法 ； 生醫奈米

並列摘要

A malignant neoplasm is abnormal cell growth in a tissue and affect human quality of life and lifespan. Lung cancer was the second most commonly estimated cancer case and the first leading cause of cancer death both in human. Therefore, lung cancer brought out over one million death worldwide each year. However, in the era of medical technology developed, the five-year relative survival rates of lung cancer, especially in advanced stage, would not significantly improve. Various chemotherapeutic drugs, such as cisplatin, were widely accepted as a standard first-line treatment for advanced NSCLC (non-small cell lung cancer). However, cisplatin performs excellent treatment, but the drug resistance may be the major obstacle to affect the treatment.
This study proposed a micro-emulsion method to synthesize cisplatin-loaded magnetite-hydroxyapatite nanoparticles (mHAp/CDDP) to combine chemotherapy and hyperthermia in one nanoparticle system for lung cancer therapy. Magnetite can act as a thermal seed under an alternating magnetic field for hyperthermia. The loading efficiency of the cisplatin in the synthesized nanoparticles was 22.9%. The major phase of the synthesized mHAP/CDDP nanoparticle was identified as hydroxyapatite by XRD; with where the magnetite and CDDP crystal were precipitated on the surface of hydroxyapatite along [211] and [200], respectively. The individual grain size of synthetic nanoparticles were in the range of 50-80 nm directly under TEM examination. The magnetic particles revealed a superparamagnetic property by SQUID; that could induce heat generation within minutes by an external alternating magnetic field and can be used for hyperthermia application. Moreover, the synthesized nanoparticle can be engulfed by A549 cells through endocytosis process. Therefore, hydroxyapatite can be dissolved in endosome-lysosome hybrid which would be breakdown by osmotic pressure and then cisplatin would escape from the vesicles into nucleus leading A549 cells toward apoptosis via ERK signaling pathway. Based on the results of in vitro and in vivo studies, to combine hyperthermia and chemotherapy in mHAp/CDDP nanoparticle could provide synergistic effect to inhibit A549 proliferation and tumor growth. We believed that the mHAp/CDDP nanoparticles have great potential on lung cancer treatment.

並列關鍵字

Lung cancer therapy ； hyperthermia ； hydroxyapatite ； microemulsion ； Nanomedicine

參考文獻

[10] Manegold C. Non-Small Cell Lung Cancer Treatment: UNI-MED Verlag AG; 2007.

[1] Ferlay J, Shin H-R, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International Journal of Cancer. 2010;127:2893-917.

[2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: A Cancer Journal for Clinicians. 2013;63:11-30.

[3] Pao W, Girard N. New driver mutations in non-small-cell lung cancer. The lancet oncology. 2011;12:175-80.

[4] Subramanian J, Govindan R. Lung cancer in never smokers: a review. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2007;25:561-70.