多功能磁性奈米粒子於大白鼠頸動脈血栓標定與藥物輸送之研究

Study of the multifunctional magnetic nanoparticle for thrombosis targeting in carotid artery in rat and drug delivery

葉日兆(Jih-Chao Yeh)

指導教授 ： 婁世亮

中原大學/工學院/生物醫學工程研究所/博士(2014年)

https://doi.org/10.6840/cycu201400406

摘要

血栓是因血管內皮細胞功能受損引起的擴散性病理過程，造成冠狀動脈疾病、腦血管疾病與短暫性腦缺血發作。此外，因一氧化氮引起的慢性發炎也會造成內皮細胞受損，進而使其失去原有抗凝血、抗血小板聚集及血管擴張的功能，導致有血栓形成的趨勢。陳皮素具有抗氧化及抗發炎的特性，然而，陳皮素在水中的低溶解度影響其對抗發炎治療的結果。另外，血管壁內皮細胞受損的位置無法早期的被診斷出來，因此，需要一個新的方法來解決上述的問題。
目前已有不同形式的藥物載體發展成為藥物輸送的方法，其中同時具有疏水性內核與親水性外殼結構的溫度應答型微胞是熱門的選擇。磁性奈米粒子可被使用為具診斷功能的磁振造影對比劑。因此，本研究將結合抗體、磁性奈米粒子及溫度應答型高分子的特性，設計一個多功能磁性奈米粒子提供藥物輸送與磁振造影時標定血栓使用。並且，為了驗證多功能磁性奈米粒子主動標定於內皮細胞受損位置的能力，本論文將開發新的血栓動物模型。
首先，本研究提出一個使用可植入式無線發光二極體裝置誘發大鼠頸動脈形成血栓動物模型的方法。此裝置由負責無線傳輸電力的體外控制器與體內發光二極體組件所組成。在動物體內注入rose Bengal後，體內植入的發光二極體組件獲得電力後，發光照射頸動脈形成血栓。使用小動物超音波、7T磁振造影及組織學染色觀察血栓形成的變化。研究結果發現，使用光功率為6 mW/cm2的發光二極體單次照射頸動脈4小時，能夠得到急性栓塞的結果；另外，使用光功率4.5 mW/cm2的發光二極體照射頸動脈2小時，關閉30分鐘，再次照射2小時，發現血栓會逐漸累積，並且於照射後第7天頸動脈會完全栓塞。與對側沒有發光二極體照射的動脈比較，發現經由發光二極體照射後的血管內皮細胞呈現不連續受損的情形。因此，透過調整發光二極體照射的時間與調整發光二極體的光功率，將能夠產生急性與漸進性血栓形成模型。此模型將能夠提供驗證多功能奈米粒子主動標定內皮細胞受損位置的能力。
第二部分，本研究提出一個製備由聚琥珀醯胺與聚異丙基丙烯醯胺及二甲基丙烯醯胺所組成之具有生物相容性與溫度應答型共聚物作為藥物載體的方法，並且探討此共聚物的相關特性。此共聚物是由帶有胺基的聚異丙基丙烯醯胺及二甲基丙烯醯胺的聚合物，透過與聚琥珀醯胺的親核開環反應所製成。此聚合物的低臨界溶液溫度為高於人體體溫的42.6 oC，外型為圓球狀且直徑為85 nm。經由細胞毒性與溶血試驗結果得到此聚合物具有相當好的生物相容性。此外，此聚合物能夠將陳皮素包覆於內核中，包覆率約為20%。經由體外藥物釋放試驗結果發現，此聚合物具有透過溫度應答控制藥物釋放的能力。與高於低臨界溶液溫度的陳皮素釋放結果比較下，僅有少部分陳皮素在低於低臨界溶液溫度的狀況下被釋放出來。藉由脂多醣體誘發一氧化氮的抑制實驗結果發現，包覆有陳皮素的共聚物能夠明顯抑制一氧化氮的生成。從以上結果得知，此具有生物相容性的溫度應答型共聚物具有作為藥物載體的能力。
最後，結合磁性奈米粒子、抗體與溫度應答型共聚物的特性，設計一個具有主動標定血栓形成位置的主動標定多功能磁性奈米粒子作為血栓診斷的工具。此主動標定多功能磁性奈米粒子是由帶胺基的磁性奈米粒子與溫度應答高分子交聯結合後，再與血管細胞黏附因子抗體(VCAM-1)交聯反應而成。此接枝有血管細胞黏附因子的多功能磁性奈米粒子仍具有直徑約600nm的圓球外型。帶胺基的磁性奈米粒子沒有明顯的細胞毒性，表示此多功能磁性奈米粒子能夠於生物醫學上使用。將接枝有血管細胞黏附因子的多功能磁性奈米粒子靜脈注射進入體內5分鐘後，能夠藉由磁振造影觀察粒子主動標定血管內皮細胞受損的位置。因此，接枝有血管細胞黏附因子的多功能磁性奈米粒子能夠作為磁振造影的顯影劑，並且及早標定血管內皮細胞受損的位置。

關鍵字

生物降解性 ； 多功能磁性奈米粒子 ； 溫度應答型高分子 ； 磁振造影顯影劑 ； LED誘發血栓模型 ； 生物相容性 ； 血栓形成 ； 藥物釋放

並列摘要

Thrombosis is a diffuse pathologic process that starts with endothelial dysfunction and clinically manifests as coronary artery disease, cerebrovascular disease, and transient ischemic attack. In addition, chronic inflammation induced by nitric oxide may also cause endothelial damage, resulting in losing the physiologic anticoagulant, antiaggregant, and vasodilatory properties of endothelium and causing thrombotic tendencies. Hesperetin is a flavanone with a wide range of antioxidant and anti-inflammatory properties. However, the poor solubility of hesperetin in aqueous buffers can cause bioavailability problems in anti-inflammatory treatments. Moreover, physicians encountered problems in the early diagnosis of the location of endothelial dysfunction on the vascular wall. Therefore, a new approach is needed to overcome the abovementioned problems.
Several types of drug carriers have been investigated. Thermoresponsive core shell-type polymeric micelles with a hydrophobic inner core and hydrophilic outer shell architecture are popular candidates. Magnetic nanoparticles can be used as magnetic resonance imaging contrast agents for diagnostic purposes. This dissertation combines the properties of antibodies, magnetic nanoparticles, and thermoresponsive copolymers to design multifunctional magnetic nanoparticles for drug delivery and thrombosis labeling in MR imaging. Furthermore, to test whether multifunctional magnetic nanoparticles can actively target endothelial injury sites, a novel thrombosis animal model was developed.
First, a novel approach to form the common carotid artery (CCA) thrombus in rats with a wireless implantable light-emitting diode (LED) device was developed. The device mainly comprises an external controller that is responsible for wirelessly transmitting electrical power and an internal LED assembly. In addition to rose Bengal dye injection into animal circulation, the internal LED assembly served as the implant that receives power and irradiates light on CCA. Thrombus formation was identified with animal sonography, 7T magnetic resonance imaging, and histopathologic examination. The present study showed that an LED assembly implanted on the outer surface of CCA could induce acute occlusion with single irradiation by a 6 mW/cm2 LED for 4 h. If intermittent irradiation with a 4.3–4.5 mW/cm2 LED for 2 h was shut off for 30 min and reapplied for another 2 h, the thrombus was observed to gradually grow and was totally occluded after 7 days. Compared with the contralateral CCA without LED irradiation, the arterial endothelium in the LED-irradiated artery was discontinued. This study has shown that by adjusting the duration of irradiation and the power intensity of LED, it is possible to produce acute occlusion and progressive thrombosis for studying the feasibility of multifunctional magnetic nanoparticles for actively labeling endothelial injury sites.
A synthesis method of biocompatible and thermoresponsive copolymers comprising poly(succinimide)-g-poly(N-isopropylacrylamide-co-N,N-dimethyl acrylamide)(PSI-g-poly(NIPAAm-co-DMAAm) as new drug carriers is provided, and their characteristics are investigated. The PSI-g-poly(PNIPAAm-co-DMAAm) copolymers were prepared by nucleophilic opening of poly(succinimide) (PSI) using amino-terminated poly(NIPAAm-co-DMAAm). The lower critical solution temperature (LCST) of the copolymer was 42.6 °C higher than the normal human body temperature. Blank copolymers were observed to have regular spherical shape with a particle diameter of approximately 85 nm. This copolymer exhibited no significant cytotoxicity and hemolysis indicated that the copolymer had good biocompatibility. In addition, these copolymers could encapsulate the anti-inflammatory hesperetin in their inner core with a drug load of approximately 20%. The release profiles of hesperetin showed significant thermoresponsive switching behavior. The hesperetin release response was dramatically lower at temperatures below LCST compared with temperatures above LCST. Lipopolysaccharide-induced nitric oxide (NO) production inhibition experiments demonstrated that hesperetin-encapsulated micelles were significantly reduced. Biocompatible thermoresponsive copolymers based on PSI-g-poly(NIPAAm-co-DMAAm) clearly have great potential as drug delivery agents.
Finally, combining the properties of antibody, magnetic nanoparticles, and thermoresponsive copolymers, active targeting multifunctional magnetic nanoparticles were designed to use as diagnostic tools for thrombosis labeling. Nanoparticles were prepared by cross-linking with PSI-g-poly(PNIPAAm-co-DMAAm) and amino-terminated magnetic nanoparticles; then, the vascular cell adhesion molecule-1 (VCAM-1) was conjugated with Fe3O4/PSI-g-poly(PNIPAAm-co-DMAAm) magnetic nanoparticles. VCAM-1-conjugated multifunctional magnetic nanoparticles have regular spherical shape with a particle diameter of approximately 600 nm. The amino-terminated magnetic nanoparticles exhibited no significant cytotoxicity, indicating that the multifunctional magnetic nanoparticles have good biocompatibility in biomedical applications. Endothelial injury sites could be actively labeled by VCAM-1-conjugated multifunctional magnetic nanoparticles and observed using magnetic resonance imaging after intravenous administration within 5 min. Thus, VCAM-1-conjugated multifunctional magnetic nanoparticles can be used as tools for the early diagnosis of the location of endothelial dysfunction on vascular walls.

並列關鍵字

thermo-responsive copolymers ； drug delivery ； MRI contract agents ； multifunctional magnetic nanoparticles ； LED induced thrombosis model ； thrombosis ； biodegradability ； biocompatibility

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