顆粒性白血球集落刺激因子(granulocyte colony-stimulating factor, GCSF)被廣泛用於治療罹患嗜中性白血球缺乏症(neutropenia)之病患。目前GCSF主要以皮下注射或是靜脈注射來投遞，而為使GCSF在體內維持一定濃度及藥效，須定時針劑給藥，對於需長期施打的病患不僅不方便亦帶來不適感。
口服投藥對於病患來說，是一個最方便且接受度高的投藥方式。但在被人體所吸收前腸胃道中的胃酸及消化酵素會降解這些蛋白質藥物，且蛋白質藥物普遍為親水性大分子，不易穿透過小腸上皮細胞，使蛋白質藥物可被小腸吸收的量相當低。為了促進小腸對蛋白質藥物的吸收，本研究利用金屬螯合劑DTPA (diethylene triamine pentaacetic acid)修飾聚麩胺酸(γ-PGA)，再與幾丁聚醣(chitosan, CS)以離子鍵結形成多功能奈米微粒做為GCSF的口服釋放載體。CS具有增加細胞間隙滲透性(paracellular permeation enhancement)的功能；DTPA可藉由螯合腸道中的二價金屬離子，來抑制酵素對GCSF所造成的降解。因此，由CS及γPGA-DTPA所形成的多功能奈米微粒具有可抑制腸道酵素對GCSF的降解與促進腸道對GCSF的吸收等功能，預期能增加口服投遞GCSF的藥物生體可用率(bioavailability)與增加嗜中性白血球的增生。
本研究主要分成四大部分。第一部分為GCSF奈米微粒的製備及特性分析，量測GCSF在奈米微粒中的包覆率，及觀察奈米微粒的粒徑及表面電荷在不同pH值環境中的變化情形。第二部分為GCSF奈米微粒多功能性評估，分別藉由小腸酵素抑制試驗與跨模電阻試驗來分析奈米微粒的功能性。第三部分是利用小鼠造血細胞NFS-60作為in vitro之模型，探討包覆於奈米微粒中的GCSF促進造血細胞增生的活性與機制探討。第四部份則是大鼠的in vivo實驗，將製備好的GCSF奈米微粒經口服投遞予大鼠後，探討其藥效學及藥物動力學之變化，並分別使用SPECT/CT與PET觀察其生物分布與治療效果。由上述實驗結果證實，此多功能奈米微粒相當有潛力做為GCSF的口服釋放載體。

Granulocyte colony-stimulating factor (G-CSF) is used to treat neutropenia patients. Although subcutaneous (SC) injections of GCSF are the conventional means to used, the inconvenience, pain, and side effects associated with such injections reduce patient compliance.
Oral administration of GCSF is a viable alternative to SC injections, since patients are more receptive to it. Nevertheless, delivering GCSF orally often leads to a low bioavailability owing to its presystemic degradation and inadequate permeation through the intestinal epithelium. To improve the oral bioavailability of GCSF, we described a pH-responsive nanoparticle system composed of chitosan (CS) and DTPA (diethylene triamine pentaacetic acid) modified poly(g-glutamic acid) (γPGA-DTPA). CS, a mucoadhesive polycation, can enhance the intestinal paracellular permeability by transiently opening the tight junctions (TJs) between epithelial cells; DTPA, which can inhibit enzymes degradation by chelating metal cations (such as Ca2+ or Zn2+).
The functional nanoparticles composed of CS and γPGA-DTPA has a mucosal adsorption, intestinal enzymes inhibition, and enhancing intestinal absorption of G-CSF, which can be expected to increase drug bioavailability.

致謝 I
摘要 II
圖目錄 V
表目錄 VI
第一章 緒論 1
1.1 嗜中性白血球缺乏症(Neutropenia) 1
1.2 顆粒性白血球集落刺激因子(GCSF) 2
1.3 口服傳遞系統(Oral delivery) 3
1.4 由幾丁聚醣(chitosan, CS)及金屬螯合劑修飾聚麩胺酸(poly(g-glutamic acid)-DTPA, γPGA-DTPA)所構成之奈米微粒 5
1.5 研究動機與目的 6
第二章 材料與實驗方法 10
2.1 小分子量幾丁聚醣之合成 10
2.2 γPGA-DTPA複合物之合成 11
2.2.1 合成γPGA-(N-Boc-hexanediamine) 11
2.2.2 合成γPGA-hexanediamine 11
2.2.3 合成γPGA-DTPA 11
2.3 包覆GCSF之多功能奈米微粒之製備與特性分析 13
2.3.1 包覆GCSF之多功能奈米微粒之製備 13
2.3.2 包覆GCSF之多功能奈米微粒之特性分析 13
2.4 包覆GCSF之多功能奈米微粒之功能性評估 14
2.4.1 抑制酵素活性分析 14
2.4.2 Transepithelial electrical resistance (TEER)量測與滲透性測試(Transport assay) 15
2.5 體外實驗(In vitro) 17
2.5.1 GCSF活性分析 17
2.5.2 體外實驗機制探討 17
2.6 體內實驗(In vivo) 18
2.6.1 藥物動力學(Pharmacokinetics, PK)之探討 18
2.6.2 藥效學(Pharmacodynamics, PD)之探討 18
2.6.3 生物分布 19
第三章 實驗結果與討論 21
3.1 包覆GCSF之多功能奈米微粒之特性分析 21
3.2 包覆GCSF之多功能奈米微粒之功能性評估 22
3.2.1 抑制酵素活性分析 22
3.2.2 Transepithelial electrical resistance (TEER)量測與滲透性測試(Transport assay) 23
3.3 體外實驗(In vitro) 25
3.3.1 GCSF活性分析 26
3.3.2 GCSF體外機制探討 27
3.4 體內實驗(In vivo) 27
3.4.1 藥物動力學(Pharmacokinetics)之探討 27
3.4.2 藥效學(Pharmacodynamics)之探討 29
3.4.3 生物分布 31
第四章 結論 35
參考文獻 36

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