研發應用於腦疾病治療之標靶型奈米藥物傳輸系統及超音波微氣泡

Development of Targeting Nano Drug Delivery Systems and Ultrasound Microbubbles for Brain Disease Treatment

陳永竹(Yung-Chu Chen)

指導教授 ： 林文澧

共同指導教授 ： 謝文元

國立臺灣大學/工學院/醫學工程學研究所/博士(2016年)

https://doi.org/10.6342/NTU201603631

摘要

血腦屏障的緊密連接構造與排外運輸系統大幅限制了95%中樞神經疾病的藥物/基因。為了促進大腦專一性的藥物傳輸，在本研究的第一部分，我們開發一種標靶型載體系統，此系統包含鍵結去辛醯化生長素釋質的poly(carboxyl ethylene glycol-g-glutamate)-co-poly(distearin-g-glutamate) (CPEGGM-PDSGM)之高分子囊胞。根據報導指出去辛醯化生長素釋質能單向通過血腦屏障 (血液通往大腦的方向)。然而，沒有任何的報導去確認去辛醯化生長素釋質鍵結在藥物載體系統上是否能藉由去辛醯化生長素釋質結合位置的介導內吞作用來標靶血腦屏障。為了定性和定量研究這種載體的特性，coumarin 6、 Cy5.5和met-enkephalin各自被包覆於高分子囊泡中。研究結果顯示當共同培養於血腦屏障細胞時，去辛醯化生長素釋質鍵結後的高分子囊胞(GPs)的細胞攝取，顯著的比未鍵結的高分子囊胞高。除此之外，在動物實驗的研究中也指出GPs對腦具有較高的選擇性，能增加在腦中的藥物累積量並減少在肝臟與脾臟的藥物累積。在熱板試驗與福馬林研究顯示，以靜脈注射包覆有met-enkephalin的GPs對於疼痛反應具有比較顯著的抑制效果。由整體結果證實，GPs具有潛力能發展通過血腦屏障並治療中樞神經性疾病的標的傳輸系統。
腦腫瘤在臨床醫學治療上一直是一個很大的挑戰，起因於無法讓足夠的藥物通過血腦屏障與藥物進入腫瘤具有低滲透性。為了有效的治療腦腫瘤與降低正常組織的副作用，因此，在研究的第二部分我們製備去辛醯化生長素釋質與葉酸鍵結的doxorubicin的高分子囊胞(GFP-D)，去促進高分子囊胞通過血腦屏障並標的腦腫瘤。尺寸測量結果顯示，此這種穿透血腦屏障與癌細胞標的型高分子囊胞的顆粒大小為約85 nm。在體外用血腦屏障細胞模型和C6膠質瘤細胞實驗結果指出，GFP-D具有較強的穿透與標靶特性的藥物傳遞。在C6細胞生存能力測試中，GFP-D比未改質的doxorubicin高分子囊泡（PD）具有較顯著的抑制效果。在體內抗腫瘤實驗中顯示，GFP-D比施予doxorubicin (Dox), doxorubicin的微脂體(L-D), P-D, 或是單一配體鍵結的高分子囊胞在抗神經膠質瘤的效果與植入腦瘤的小鼠總體存活率方面都較高。此外， Cy5.5也被包覆用來評估此種穿透性標靶傳輸系統的遞送特性。整體的實驗結果指出，此種穿透性標靶傳輸系統GFP-D在腦腫瘤的治療上是一種具有高潛力的奈米載體。
在本研究的第三部分，我們開發去辛醯化生長素釋質鍵結的微氣泡(GMB)並包覆TGFβ1抑制劑 (LY364947) (GMBL)，並藉由結合超音波(US)的方式誘導血腦屏障/血瘤屏障(BBB / BTB)的瓦解。在體外穩定性研究中顯示，GMB的穩定性能維持一個月以上。在體內研究中也指出，施予folate鍵結的doxorubicin 高分子囊胞 (FPD) 並經由 GMBL/US 處理(FPD+GMBL/US)，能達到最好的抑制神經膠質瘤的效果並能改善植入腦瘤的小鼠總體存活率。當結合聚焦是超音波時，GMBL能促進血腦屏障/血瘤屏障的瓦解並同時釋放出LY364947，LY364947能降低腦腫瘤內皮的週細胞覆蓋，會增強FPD的累積和抗腫瘤活性。整體的實驗結果指出，FPD+GMBL/US這種非侵入性標靶傳輸方式，能促進奈米藥物進入到腦腫瘤中，並達到治療的效果與降低正常腦組織的藥物毒性，此種治療方式於CNS疾病治療上是具有很大的潛力。

關鍵字

高分子囊胞 ； 微氣泡 ； TGFβ1抑制劑 ； 葉酸 ； 血腦/血瘤屏障（BBB/ BTB) ； 標靶傳輸 ； 超音波

並列摘要

The effective protection of the blood-brain barrier (BBB) from tight junctions and efflux transport systems ultimately results in the limited entry of 95% of drug/gene candidates, which are potentially beneficial for central nervous system (CNS) diseases. In order to enhance the brain-specific delivery, in present part of this study we developed a targeting nano drug carrier system, which consists of poly(carboxyl ethylene glycol-g-glutamate)-co-poly(distearin-g-glutamate) (CPEGGM-PDSGM) polymersomes with the conjugation of des-octanoyl ghrelin. Des-octanoyl ghrelin across the BBB was reported to be unidirectional (blood-to-brain direction). However, there is no report about the conjugation of des-octanoyl ghrelin to a drug carrier system to confer the BBB targeting property through des-octanoyl ghrelin binding sites mediated endocytosis. To qualitatively and quantitatively investigate this carrier’s properties, coumarin 6, Cy5.5 and met-enkephalin were individually encapsulated in these polymersomes. The experimental results showed that the cellular uptake was significantly higher for des-octanoyl ghrelin-conjugated polymersomes (GPs) than unconjugated polymersomes when co-incubated with the BBB cells. In addition, an enhanced accumulation in brain together with a reduced accumulation in liver and spleen was observed in animal study, indicating better brain selectivity for the GPs. In a hot-plate test and formalin test, a significant inhibition of nociceptive response could be achieved for an intravenous injection of GPs encapsulated with metenkephalin. The overall results demonstrated that GPs own a great potential for targeting delivery of drug across the BBB to treat CNS diseases.
Chemotherapy for brain cancer tumors remains a big challenge for clinical medicine due to the inability to transport sufficient drug across the BBB and the poor penetration of drug into the tumors. To effectively treat brain tumors and reduce side effects on normal tissues, both des-octanoyl ghrelin and folate conjugated with polymersomal doxorubicin (GFP-D) was developed in second part of this study to help transport across the BBB and target the tumor as well. The size measurements revealed that this BBB-penetrating cancer cell-targeting GFP-D was about 85 nm. In-vitro experiments with a BBB model and C6 glioma cells demonstrated that GFP-D owned a robust penetrating-targeting function for drug delivery. In C6 cell viability tests, GFP-D exhibited an inhibitory effect significantly different from the unmodified polymersomal doxorubicin (P-D). In-vivo antitumor experiments showed that GFP-D performed a much better anti-glioma effect and presented a significant improvement in the overall survival of the tumor-bearing mice as compared to the treatments with free doxorubicin (Dox), liposomal doxorubicin (L-D), P-D, or single ligand conjugated P-D. In addition, Cy5.5 was used as a probe to investigate the delivery property of this penetrating-targeting delivery system. The overall experimental results indicate that this BBB-penetrating cancer cell-targeting GFP is a highly potential nanocarrier for the treatment of brain tumors.
In third part of this study, we developed the des-octanoyl ghrelin conjugated microbubbles (GMB) loaded with TGFβ1 inhibitor (LY364947) (GMBL) to induce BBB/BTB disruption for ultrasound (US) sonication with GMBL. The in-vitro stability study showed that GMB was pretty stable over one month. The in-vivo study showed that the combination of folate-conjugated polymersomal doxorubicin (FPD) and GMBL/US (FPD+GMBL/US) achieved the best anti-glioma effect and significant improvement in the overall survival time for brain tumor-bearing mice. When combined with focused US, GMBL facilitated local BBB/BTB disruption and simultaneously released LY364947 to decrease the pericyte coverage of the endothelium at the targeted brain tumor sites, resulting in enhanced accumulation and antitumor activity of FPD. The overall results indicate that GMBL/US owns a great potential for non-invasive targeting delivery of nanomedicine across the BBB to treat central nervous system (CNS) diseases.

並列關鍵字

polymersomes ； microbubbles ； TGFβ1 inhibitor ； folate ； blood-brain/blood-tumor barrier (BBB/BTB) ； targeted delivery ； ultrasound

參考文獻

[1] Shoichet MS, Winn SR. Cell delivery to the central nervous system. Adv Drug Deliv Rev 2000;42:81–102.

[2] Ngwuluka N, Pillay V, Du Toit LC. Levodopa delivery systems: Advancements in delivery of the gold standard. Expert Opin Drug Deliv 2010;7:203–224.

[3] Pathan SA, Iqbal Z, and Zaidi SM. CNS drug delivery systems: Novel approaches. Recent Pat Drug Deliv Formul 2009;3:71–89.

[4] Ricci M, Blasi P, Giovagnoli S, and Rossi C. Delivering drugs to the central nervous system: A medicinal chemistry or a pharmaceutical technology issue? Curr Med Chem 2006;13:1757–1775.

[6] Pardridge WM. Blood-brain barrier delivery. Drug Discov Today 2007;12:54–61.