臺灣博碩士論文加值系統

重組體組織漿胞素原活化劑(recombinant tissue-type plasminogen activator; rtPA)被用於治療血栓性疾病，然而在臨床使用上有嚴重的出血性副作用，又因循環中的內生性抑制劑導致半衰期短。過去研究顯示固定於奈米載體表面的rtPA可於動脈給藥及磁導時引發標靶溶栓效應，且因所需劑量減少而有望減少其副作用。此計畫測試以下假說:靜脈給予包覆型rtPA以免於內生性抑制劑作用時可控制藥物於局部釋放引發標靶性溶栓。自發釋放與控制釋放型的rtPA 分別以幾丁聚醣包覆奈米複合材料(MCNP@rtPA)與溫感型磁性奈米微脂體(TML@rtPA)由本校陳志平教授實驗室製備及優化後於體外和體內進行測試。藉由血栓彈性分析系統測試，MCNP@rtPA顯著降低溶解指數(lysis index, LI)，但對凝血時間(coagulation time, CT)並無影響。與37°C組別比較，於43°C預熱的TML@rtPA更進一步降低LI，顯示TML@rtPA釋放rtPA呈現溫度依賴性。利用磁場導引策略於大鼠血栓栓塞模型中，動脈給予MCNP@rtPA (20% rtPA常規劑量) 後引發髂動脈血流顯著回復；藉溫度控制系統使局部維持43°C時，靜脈遠端給予TML@rtPA亦可使髂動脈血流顯著回復，但回復速度較動脈給藥時慢。因此，保護型rtPA劑型與合適的標靶策略對大鼠血栓栓塞模型中由靜脈給予rtPA奈米藥物以達到有效標靶性溶栓是極為重要的。

Recombinant tissue-type plasminogen activator (rtPA) is approved as a thrombolytic agent for treatment of thromboembolic diseases; however, short half-life due to endogenous inhibitors in circulation and severe hemorrhagic effects are observed in clinical use. Our previous studies indicated that intra-arterial (i.a.) administration of immobilized rtPA under magnetic guiding induces target thrombolysis. Target delivery of such nanocomposites requires smaller dose, which may reduce its adverse effects in application. In this project, we hypothesized that i.v. administration of encapsulated rtPA, which is protected from endogenous inhibitors, may induce target thrombolysis following controlled-release of rtPA. Spontaneous vs. controlled release of rtPA from chitosan-coated magnetic nanocomposites (MCNP@rtPA) vs. thermosensitive magnetoliposomes (TML@rtPA), respectively, were prepared and optimized by Professor Jyh-Ping Chen’s Lab, and tested both in vitro and in vivo. With thromboelastomety, MCNP@rtPA significantly lowered lysis index (LI), but no effect was observed on coagulation time (CT). Preincubation of TML@rtPA at 43°C further reduced LI compared to that at 37°C, suggesting a thermo-dependent rtPA release from TML@rtPA. In a rat embolic model with magnetic guiding strategy, i.a. MCNP@rtPA with 20% of rtPA regular dose induced significant restore of iliac blood flow (IBF). At focal site of 43°C with a thermal controlled system, i.v. TML@rtPA also induced significant IBF restore, which occurred slower than that induced by i.a. TML@rtPA. In conclusion, both protected rtPA and a targeting strategy are imperative for target thrombolysis in response to i.v. administration of rtPA nanocomposites in the rat embolic model.

Recommendation Letter from the Thesis Advisor…………………………….
Thesis/Dissertation Oral Defense Committee Certification…………………..
誌謝 iii
摘要 iv
Abstract v
Table of Contents vi
List of Figures viii
List of Tables x
Abbreviation xi
1. Introduction 1
1.1 Plasminogen activators 1
1.1.1 Tissue-type plasminogen activators 2
1.1.2 Urokinase-type plasminogen activators 2
1.1.3 Adverse effects of plasminogen activators 3
1.2 Target delivery of plasminogen activators 4
1.2.1 Magnetic targeting 4
1.2.2 Ligand targeting 5
1.3 Consideration of polymer coating of nanocarriers 6
1.4 Endogenous inhibitors of rtPA 7
1.5 Encapsulated rtPA 8
1.6 Controlled release of rtPA 10
2. Materials and Methods 13
2.1 Materials 13
2.2 In vitro chromogenic assay of rtPA activity 14
2.3 Thromboelastometry 14
2.4 A rat embolic model 15
2.5 The thermo-controlled system 17
2.6 MRI analysis 17
2.7 Tunable Resistive Pulse Sensing (TRPS) 18
2.8 Statistical analysis 18
3. Results 19
3.1 In vitro thrombolysis of MGO-rtPA 19
3.2 In vitro thrombolysis of MCNP@rtPA 20
3.3 In vitro thrombolysis of TML@rtPA at 30°C vs. 40°C 21
3.4 In vitro thrombolysis of TML@rtPA preincubated at 37°C vs. 43°C 22
3.5 Effects of PLGA-MNP-rtPA/uPA and MGO-rtPA in vivo 22
3.6 MCNP@rtPA-induced thrombolysis in vivo 23
3.7 TML@rtPA-induced thrombolysis in vivo 24
3.8 Magnetic Resonance Imaging analysis 26
4. Discussion 28
4.1 In vitro thrombolysis 28
4.2 The target thrombolysis models 28
4.3 MCNP@rtPA 29
4.4 TML@rtPA 32
5. Conclusion 38
6. Future Perspective 39
References 42
Tables 52
Figures 54
Appendix 73

List of Figures
Figure 1 Strategic plan in preparation and characterization of nanocomposites for targeted thrombolysis. 54
Figure 2 Schematic diagrams of various nanocomposites. 55
Figure 3 Concentration-dependent thrombolysis induced by MGO-rtPA in whole blood. 56
Figure 4 Concentration-dependent thrombolysis with MCNP@rtPA. 57
Figure 5 Thrombolytic effects of MCNP@rtPA vs. free rtPA. 58
Figure 6 Coagulation and thrombolysis induced by TML@rtPA at 30°C vs. 40°C. 59
Figure 7 Thermal effects of thrombolysis induced by TML@rtPA in vitro. 60
Figure 8 Temperature effects of rtPA activity at 37°C vs. 43°C. 61
Figure 9 Schematic diagram of a rat embolic model for target thrombolysis. 62
Figure 10 Application of magnetic force in target delivery of encapsulated nanocomposites in a rat embolic model. 63
Figure 11 Hemodynamic effects of intra-arterial administration of PLGA-MNP-rtPA in a rat embolic model. 64
Figure 12 Hemodynamic effects of intra-arterial administration of PLGA-MNP-uPA in a rat embolic model. 65
Figure 13 The thrombolytic effects of MGO-rtPA with or without anti-fibrin peptide in a rat embolic model. 66
Figure 14 Target thrombolysis with MCNP@rtPA in a rat embolic model. 67
Figure 15 The working hypothesis of target thrombolysis induced by thermal controlled release of rtPA from magnetoliposomes (TML@rtPA) using magnetic guidance in the artery of the rat embolic model. 68
Figure 16 The representative profiles of focal hyperthermal system. 69
Figure 17 Intra-arterial TML@rtPA-induced target thrombolysis. 70
Figure 18 Intra-venous TML@rtPA-induced target thrombolysis. 71
Figure 19 Biodistribution of MCNP as determined by MRI. 72

List of Tables
Table 1 Size distribution and surface charge of different nanocomposites in various mediums using dynamic light scattering (DLS) and tunable resistive pulse sensing (TRPS). 52
Table 2 Hematological analysis of the blood from rats administered with TML at 37°C, TML@rtPA at 37°C, TML@rtPA at 43°C and free rtPA at 43°C. 53

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