臺灣博碩士論文加值系統

本研究使用polybutylcyanoacrylate (PBCA) nanoparticles、methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) nanoparticles和solid lipid nanoparticles (SLN)為stavudine (D4T)、delavirdine (DLV)和saquinavir (SQV)載體，進行藥物對奈米粒子的負載效率(loading efficiency)及生體外(in vitro)血腦阻障(blood-brain barrier, BBB)模型的藥物釋放(drug release)研究，同時為了更接近人體狀態與符合組織工程概念，因此是以繼代培養(subculture)人腦微血管內皮細胞(human brain microvessel endothelial cells, HBMECs)來建立本模型。藥物在PBCA和MMA-SPM奈米粒子上的負載效率大小次序：D4T > DLV > SQV；D4T負載在奈米粒子上的負載效率大小次序：MMA-SPM > PBCA，DLV和SQV負載在奈米粒子上的負載效率大小次序：PBCA > MMA-SPM；藥物負載在PBCA和MMA-SPM奈米粒子上的負載效率隨奈米粒子粒徑增大而減少。SLN包覆率大小次序：SQV > DLV > D4T，SLN粒徑隨包覆率增加而變大。單純藥物穿透率大小次序：D4T > SQV > DLV；一般而言在穿透提升率大小次序：PBCA > SLN>MMA-SPM，藥物負載在PBCA和MMA-SPM奈米粒子上的穿透率隨奈米粒子粒徑增大而降低；藥物包覆在SLN內隨粒子粒徑增大而降低。此外PBCA和MMA-SPM奈米粒子在同一粒徑下負載效率隨載體量增加而增加，但不影響BBB穿透率；同時使用tripalmitin和cacao butter在不同比例下為中間相合成不同粒徑之SLN，其中DLV和SQV隨cacao butter量增加包覆率下降，D4T則反之，並於4 ℃環境下儲存六週後粒子穩定度良好。

Transport study of stavudine (D4T), delavirdine (DLV) and saquinavir (SQV) by polybutylcyanoacrylate (PBCA) nanoparticles (NPs), methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) nanoparticles and solid lipid nanoparticles (SLN) across the in vitro blood brain barrier (BBB) model system was presented and investigation loading efficiency (LE) of there the durgs onto the three NPs. At the same time for similar human state and accorder with the tissue engineering concept, through human brain microvessel endothelial cells (HBMECs) were cell lines by subculture technique. The drugs LE on PBCA and MMA-SPM series : D4T > DLV > SQV; D4T loaded on NPs series : MMA-SPM > PBCA, DLV and SQV loaded on NPs : PBCA > MMA-SPM; LE decreased following by the diameter of PBCA and MMA-SPM NPs increased. The drugs entrapment efficiency (EE) in SLN series : SQV > DLV > D4T, SLN of diameter enhanced following by the EE. The drugs permeabilities series: D4T > SQV > DLV; the drugs permeabilities enhanced across the BBB by NPs series: PBCA > SLN>MMA-SPM, permeabilities decreased following by the diameter of PBCA and MMA-SPM NPs increased ; SLN is opposite. In addition, PBCA and MMA-SPM under the same diameter LE enhanced following by the carrier amount increase , but does not influence the BBB permeability; Use different Proportion of tripalmitin and cacao butter formate as internal phase synthesized SLN, DLV and SQV EE decreased following by cacao butter amount increased, D4T on the contrary, and store the steady degree of particle after six weeks well under 4 degrees Centigrade of environment.

中文摘要………………………………………………………........Ι
英文摘要…………………………………………………………..Ш
目錄………………………………………………………………..V
圖目錄…………………………………………………………......ΙΧ
表目錄……………………………………………………………ΧП
第一章 緒論
1.1愛滋病與人類免疫不全逆轉錄病毒…………………………..1
1.2藥物載體………………………………………………………..1
1.3 研究動機與目的…………………………………………….....3
第二章 文獻回顧
2.1 血腦阻障…………………………...…………………………..6
2.2 生體外血腦阻障模型………………………………………….7
2.3 Stavudine 、Delavirdine 和Saquinavir藥物簡介…………....9
2.3.1 Stavudine…………………………………………………...9
2.3.2 Delavirdine………………………………………………..10
2.3.3 Saquinavir…………………………………………………11
2.4藥物載體與血腦阻障………………………………………….12
2.4.1 PBCA奈米粒子…………………………………………..12
2.4.2 MMA-SPM奈米粒子…………………………………….14
2.4.3 SLN………………………………………………………..15
2.5表面電荷和親疏性與血腦阻障……………………………….16
2.6電磁場與血腦阻障…………………………………………….18
第三章 實驗材料、儀器、原理及方法
3.1 實驗材料……………………………………………………...22
3.1.1人腦微血管內皮細胞培養PC膜表面預處理所用材料..………………………………………………………..22
3.1.2人腦微血管內皮細胞繼代培養所用材料……………….22
3.1.3人腦微血管內皮細胞保存所用材料……………………..23
3.1.4免疫螢光法所用的材料…………………………………..23
3.1.5奈米粒子合成與乾燥所用材料…………………………..23
3.1.5.1 PBCA奈米粒子合成………………………………...23
3.1.5.2 MMA-SPM copolymer奈米粒子合成………………24
3.1.5.3 SLN合成……………………………………………...24
3.1.5.4 PBCA、MMA-SPM奈米粒子和SLN的乾燥所用材料……………………………………………………..25
3.1.6 藥物的吸收波長量測所用材料…………………………25
3.1.7 抗HIV藥負載所用材料…………………………………25
3.1.8 奈米粒子表面包覆所用材料……………………………27
3.1.9抗HIV藥負載負載於奈米粒子之FT-IR分析所用材料..27
3.1.10藥物穿透實驗所用材料………………………………...27
3.1.11螢光奈米粒子與螢光SLN………………………………27
3.1.12 毒性測試與電阻測定…………………………………..28
3.1.13其他實驗器具和耗材…………………………………....28
3.2 實驗儀器……………………………………………………...30
3.3 實驗原理及方法……………………………………………...33
3.3.1人腦內皮細胞培養PC膜的表面預處理……………….....33
3.3.2 培養細胞用的培養基配置步驟…………………………33
3.3.3 冷凍液配製步驟…………………………………………34
3.3.4 Gelatin 配製步驟………………………………………...34
3.3.5 Trypsin-EDTA 配製步驟………………………………...34
3.3.6 繼代培養之培養皿預處理………………………………34
3.3.7 人腦微血管內皮細胞繼代培養…………………………35
3.3.8 人腦微血管內皮細胞培養………………………………36
3.3.8.1 人腦微血管內皮細胞培養PC膜之實驗……………36
3.3.8.2 人腦微血管內皮細胞緊密接合(tight-junction, TJ)特性的鑑定……………………………………………36
3.3.9 奈米粒子合成……………………………………………37
3.3.9.1.1 PBCA奈米粒子合成………………………………37
3.3.9.1.2 螢光PBCA奈米粒子合成………………………...38
3.3.9.2.1 MMA-SPM奈米粒子合成………………………...38
3.3.9.2.2 螢光MMA-SPM奈米粒子製備…………………..38
3.3.9.3.1 SLN合成…………………………………………...39
3.3.9.3.2 螢光SLN製備……………………………………...40
3.3.10 抗HIV藥的吸收波長量測……………………………...40
3.3.11 奈米粒子負載與包覆……………………………….….40
3.3.11.1 抗HIV藥在PBCA和MMA-SPM上負載及奈米粒子包覆………………………….……………………...40
3.3.11.2 抗HIV藥被包覆在SLN………………………….…40
3.3.11.3 以不同中間相比例合成SLN來包覆藥物................41
3.3.11.4 抗HIV藥負載於奈米粒子之FT-IR分析…………..42
3.12 抗HIV藥的生體外BBB模型穿透實驗……………………..42
3.3.12.1 D4T、DLV和SQV的穿透實驗……………….……42
3.3.12.2 抗HIV藥物負載於奈米粒子且被polysorbate 80包覆之穿透驗…………………………………………..43
3.3.12.3 包覆抗HIV藥物之SLN穿透實驗…………………43
3.3.12.4 物質穿透生體外BBB模型之穿透率係數計算…...44
3.3.12.4.1 抗HIV藥物負載於奈米粒子且被polysorbate 80包覆之穿透率計算…………………………………45
3.3.12.4.2 包覆抗HIV藥物之SLN穿透率計算…………….46
3.3.12.5 奈米粒子被腦微血管內皮細胞吸收……………...46
3.3.13 奈米粒子對細胞毒性測試與細胞電阻測定………..47

第四章 結果與討論
4.1 奈米粒子粒徑分析及鑑定…………………………………...49
4.1.1 Zeta Sizer 3000…………………………………………49
4.1.2 FE-SEM………………………………………………...49
4.1.3 AFM…………………………………………………….50
4.2 人腦微血管內皮細胞的緊密接合(tight-junction, TJ)鑑定….50
4.3 奈米粒子對D4T、DLV和SQV之負載及包覆……………51
4.3.1 D4T、DLV和SQV對各種粒徑的PBCA之負載率…51
4.3.2 D4T、DLV和SQV對各種粒徑的MMA-SPM之負載率………………………………………………………..52
4.3.3各種粒徑的SLN對D4T、DLV和SQV之包覆率…53
4.3.4兩種成份中間相以不同比例合成SLN對D4T、DLV和SQV之包覆率與冷藏穩定實驗………………………54
4.3.5 藥物與奈米粒子之紅外線光譜實驗…………………54
4.4 生體外BBB穿透實驗……………………………………….56
4.4.1 D4T、DLV和SQV之穿透實驗……………………..56
4.4.2 D4T、DLV和SQV負載於各種粒徑之不同奈米粒子穿透實驗………………………………………………….57
4.4.2.1 D4T、DLV和SQV負載於各種粒徑PBCA奈米粒子穿透實驗…………………………………………….57
4.4.2.2 D4T、DLV和SQV負載於各種粒徑MMA-SPM奈米粒子穿透實驗………………………………………..59
4.4.2.3 包覆D4T、DLV和SQV的各種粒徑SLN穿透實驗..62
4.5 奈米粒子被腦微管內皮細胞吸收實驗……………………...64
4.6 負載率與穿透率關係………………………………………...65
4.7 奈米粒子對細胞毒性測試與細胞電阻測定………………...65

第五章 結論與建議
5.1 結論...........................................................................................69
5.2 建議...........................................................................................70
參考文獻…………………………………………………………..71
附錄………………………………………………………………139

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