用於癌症治療與診斷的多功能奈米粒子

Multifunctional Nanoparticles for Cancer Theranostics

梁博欽(Po-Chin Liang)

指導教授 ： 林文澧

國立臺灣大學/工學院/醫學工程學研究所/博士(2019年)

https://doi.org/10.6342/NTU201902917

摘要

癌症是台灣最常見的死因，根據台灣衛生福利部公布的資料，2017年癌症奪走4萬8037條人命，再創歷史新高，蟬聯36年十大死因之首。化學治療是治療癌症的主要方法之一，然而傳統的化療藥物在體內非特定地分佈，它們同時影響癌症和健康細胞，導致劑量相關的副作用和到達腫瘤的藥物濃度不足。多功能奈米粒子能靶向和控制向腫瘤細胞釋放化療藥劑，從而減少藥物誘發的全身性副作用，並提高局部腫瘤治療效果。
在我們的第一項研究中，我們開發了多功能磁性奈米顆粒SPIO-PEG-D，它是由超順磁性氧化鐵 (SPIO) 磁性核心和親水性聚乙烯乙二醇 (PEG) 外殼組成，之後再鍵結Doxorubicin (Dox) 而形成SPIO-PEG-D，它同時具有腫瘤磁振成像(MRI)和腫瘤化學治療的功用。SPIO奈米粒子的大小大約10nm，可以由穿透式電子顯微鏡觀察。而由振動樣品磁力計生成的遲滯曲線，可以發現SPIO-PEG-D仍具有超順磁性，其磁性與SPIO-PEG沒有顯著差別，表示不會因為鍵結Doxorubicin 而影響其磁性。SPIO-PEG-D 的橫向鬆弛度 (r2) 明顯高於縱向鬆弛度 (r1) (r2/r1 = 9)，因此我們可以由磁振T2加權影像來觀察SPIO-PEG-D的分佈。經由將Doxorubicin結合在SPIO-PEG的表面，因為PEG的隱藏效應，可以減少Doxorubicin的降解，因此延長了Doxorubicin在血液循環中的半衰期。在體外實驗中發現，SPIO-PEG-D可導致HT-29癌細胞DNA交聯更嚴重，使其DNA表現降低，細胞凋亡升高。在普魯士藍染色研究中發現,在外加磁場中用SPIO-PEG-D治療的腫瘤，其腫瘤內鐵密度遠遠高於單獨使用SPIO-PEG-D治療的腫瘤。在體內MRI研究中發現，有外加磁場用SPIO-PEG-D治療的腫瘤，其T2加權信號比沒有外加磁場單獨使用SPIO-PEG-D治療的腫瘤更強，顯示外加磁場可以吸引更多SPIO-PEG-D累積在腫瘤組織中。在SPIO-PEG-D的抗癌效率研究中顯示，有外加磁場組比沒有外加磁場組的腫瘤明顯縮小。在體內實驗中還發現，SPIO-PEG-D這種藥物輸送系統結合局部外加磁場可以減少心毒性和肝毒性的副作用。我們的第一項研究結果顯示,我們研製的多功能磁性奈米顆粒SPIO-PEG-D，在MRI監測及外加磁場增強腫瘤化療效果具有相當大的潛力。
在我們的第二項研究中，我們開發了多功能磁性奈米凝膠顆粒iMNP-D ，它是由iRGD鍵結溫度/pH雙敏感的磁性奈米凝膠顆粒(MNP-D)，而此凝膠顆粒中裝有超順磁性氧化鐵奈米顆粒和Doxorubicin。溫度/pH雙敏感聚合物(丙烯酸-共聚(乙二醇)二丙烯醯胺-共聚-N-異丙烯醯胺(Poly(AA-CO-PEGDA-CO-NIPAM))奈米凝膠，是通過自由基聚合而成。正電荷的Doxorubicin ，在pH7.4的環境下，經由靜電相互作用，而引入帶負電荷的奈米凝膠中。SPION 和 Doxorubicin 均封裝在奈米凝膠中，以合成溫度/pH 雙敏感磁性奈米凝膠顆粒 (MNP-D)，然後將iRGD結合在 MNP-D 表面形成 iMNP-D，iMNP-D因為與iRGD結合，因而提高其腫瘤靶向和穿透效率。因為iRGD可以與腫瘤的 intergrin 與 neuropilin-1的受體結合，而被內化進入腫瘤細胞。另外由於iMNP-D具有溫度/pH雙敏感特性，所以可以利用周圍環境溫度/pH值的變化，來改變其大小和親水/疏水性，從而控制藥物釋放。Doxorubicin 在酸性及高溫的環境下容易從 iMNP-D中釋放出來。而腫瘤的酸性環境正好有利於Doxorubicin的釋放。另外，我們可以利用短時間的局部高溫，來啟動Doxorubicin從 iMNP-D中釋放出來。我們對iMNP-D進行了增強HT-29結腸癌化療和MR成像的評估。體外和體內研究證明iRGD的存在提高了Doxorubicin對結腸癌細胞/腫瘤的細胞毒性效率,並顯示iMNP-D可以專門將Doxorubicin輸送到結腸癌。此外經由短時間局部高熱來控制藥物釋放因而促進抗腫瘤療效。
總體實驗結果顯示，這種高腫瘤穿透性，高癌細胞靶向的iMNP-D，是結腸癌的高潛力診斷與治療兼具的多功能磁性奈米凝膠載體。

關鍵字

癌症 ； 化療 ； 阿黴素 ； 內化精胺酸-甘胺酸-天門冬胺酸 ； 磁體增強 ； 磁振造影成像 ； 磁振造影監測 ； 奈米凝膠 ； 聚乙烯乙二醇 ； 超順磁性氧化鐵 ； 溫度/酸鹼敏感 ； 腫瘤

並列摘要

Cancer is the most common cause of death in Taiwan, with 48,037 people killed in 2017, a record high and the top 10 causes of death in 36 years, according to data released by Taiwan's Ministry of Health and Welfare. Chemotherapy is one of the main ways to treat cancer, while conventional cancer chemotherapeutic drugs are distributed nonspecifically in the body and hence they affect both cancerous and healthy cells, resulting in dose-related side effects and inadequate drug concentrations reaching the tumor. Multifunctional nanoparticles can target and control releasing chemotherapy agent to tumor cells, thus it can reduce drug induced systemic side effects and improve local tumor treatment results.
In the first study, we developed multifunctional magnetic nanoparticle superparamagnetic iron oxide – polyethylene glycol- Doxorubicin (SPIO-PEG-D), consisting of a superparamagnetic iron oxide (SPIO) magnetic core and a shell of aqueous stable polyethylene glycol (PEG) conjugated with doxorubicin (Dox), for tumor magnetic resonance imaging (MRI) and chemotherapy. The size of SPIO nanoparticles was ~10 nm, which was visualized by transmission electron microscope (TEM). The hysteresis curve, generated with vibrating-sample magnetometer, showed that SPIO-PEG-D was superparamagnetic with an insignificant difference as compared to superparamagnetic iron oxide – polyethylene glycol (SPIO-PEG). The transverse relaxivity (r2) for SPIO-PEG-D was significantly higher than the longitudinal relaxivity (r1) (r2/r1 = 9), so we can observe the distribution of SPIO-PEG-D by MRI T2WI. The half-life of Dox in blood circulation was prolonged by conjugating Dox on the surface of SPIO with PEG to reduce its degradation, by stealth shielding effect of PEG. The in vitro experiment showed that SPIO-PEG-D could cause DNA crosslink more serious, resulting in a lower DNA expression and a higher cell apoptosis for HT-29 cancer cells. The Prussian blue staining study showed that the tumors treated with SPIO-PEG-D under a magnetic field had a much higher intratumoral iron density than the tumors treated with SPIO-PEG-D alone. The in vivo MRI study showed that the T2-weighted signal was stronger for the group under a magnetic field, indicating that it had a better accumulation of SPIO-PEG-D in tumor tissues. In the anticancer efficiency study for SPIO-PEG-D, the results showed that there was a significantly smaller tumor size for the group with a magnetic field than the group without. The in vivo experiments also showed that this drug delivery system SPIO-PEG-D combined with a local magnetic field could reduce the side effects of cardiotoxicity and hepatotoxicity. The results showed that our developed multifunctional magnetic nanoparticle SPIO-PEG-D owns a great potential for MRI-monitoring and magnet-enhancing tumor chemotherapy.
In the second study, we developed multifunctional magnetic nanogel particle iMNP-D (iRGD-conjugated magnetic nanogel particles – Doxorubicin) , which was made of iRGD (internalized Arginine–glycine–aspartic acid) conjugated temperature/pH dually sensitive magnetic nanogel particles (MNP-D), which was loaded with superparamagnetic iron oxide nanoparticles and doxorubicin. Temperature/pH dually sensitive poly(acrylic aicd-co-poly (ethylene glycol) di-acrylamide-co-N-isopropylacrylamide (poly(AA-co-PEGDA-co-NIPAM)) nanogels were synthesized by free radical polymerization. Positively charged Dox was introduced into the negatively charged nanogels by electrostatic interaction at pH 7.4. Both Superparamagnetic iron oxide nanoparticles (SPIONs) and Dox were encapsulated in nanogels to develop temperature/pH dually sensitive magnetic nanogel particles (MNP-D), followed by conjugating the peptide iRGD on the MNP-D surface to form iMNP-D, and iMNP-D could enhance its tumor targeting and penetrating efficiency by the conjugation of iRGD. Because iRGD could be internalized into tumor cells by intergrin and neuropilin-1 receptor. Besides, the iMNP-D maintains temperature/pH dually sensitive properties and it can change its size and hydrophilic/hydrophobic properties by changing environment temperature/pH, thus achieving controlled drug release. Dox could be released from iMNP-D at a low pH and/or a high temperature. And the acid environment of tumor could trigger the release of Dox from iMNP-D. In addition, we can apply short-time local hyperthermia to trigger Dox releasing from iMNP-D. We evaluated iMNP-D for both enhancing HT-29 colon cancer chemotherapy and MR imaging. In-vitro and in-vivo studies proved that the presence of iRGD enhanced the cytotoxic efficiency of Dox to colon cancer cells/tumors and indicated that iMNP-D can deliver Dox specifically to colon cancer and can control drug release with a short-time local hyperthermia to promote anti-tumor efficacy.
The overall experimental results indicate that this high tumor-penetrating, high cancer cell-targeting iMNP-D is a highly potential theranostic multifunctional magnetic nanogel carrier for the monitoring and treatment of colon cancer.

並列關鍵字

cancer ； chemotherapy ； Doxorubicin(Dox) ； iRGD ； magnet enhancing ； MR imaging ； MRI monitoring ； nanogel ； polyethylene glycol ； superparamagnetic iron oxide ； temperature/pH sensitive ； tumor

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