新穎奈米微粒載體於口服蛋白質藥物傳遞的應用研究

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研究生：	林宥欣 Yu-Hsin Lin
論文名稱：	新穎奈米微粒載體於口服蛋白質藥物傳遞的應用研究 Novel Nanoparticles for Oral Delivery of Protein Drugs via Paracellular Pathways
指導教授：	宋信文 Hsing-Wen Sung
口試委員:
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2006
畢業學年度：	94
語文別：	英文
論文頁數：	83
中文關鍵詞：	nanoparticles 、insulin 、paracellular transport 、tight junction 、diabetes
相關次數：	點閱：2 下載：0
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An oral route is the most convenient and comfortable means of administering protein drugs, and eliminates pain caused by an injection, the stress association with multiple daily injections and possible infections. Oral administration has a higher patient compliance rate than injections. However, oral administration of protein drugs also has certain obstacles, as drugs must overcome various significant barriers in the gastrointestinal (GI) tract prior to delivery to the blood stream. First, protein drugs are rapidly degraded by the low pH of gastric medium in the stomach. Secondly, some digestive enzymes in the stomach and small intestine may lead to degradation of protein drugs. Finally, the intestinal epithelium is a major barrier to the absorption of protein drugs, as they cannot diffuse across the cells through the lipid-bilayer cell membranes.
The study presents a novel nanoparticle (NP) delivery system which is able to provide a protection from the GI environment and enhances absorption of protein drugs in the intestinal epithelium. This novel delivery system was prepared using a simple and mild ionic-gelation method to which a poly-γ-glutamic acid (□-PGA) solution was added to a low molecular-weight chitosan (low-MW CS) solution. The particle size and the zeta potential value of the prepared NPs can be controlled under various weight ratios of CS to γ-PGA. The diameters of the prepared NPs were 110-150 nm and these NPs had a negative or positive surface charge depending on the relative concentrations of CS to □-PGA utilized. X-ray diffractograms demonstrated that the crystal structure of CS was disrupted when combined with □-PGA. The ionized CS and γ-PGA formed polyelectrolyte complexes via electrostatic interactions. The FT-IR spectra showed that CS and □-PGA were ionized at pH 2.5-6.6 and the prepared NPs remained intact in the pH range of 2.5-6.8. By TEM examinations, the proposed NPs have a matrix structure and are spherical in shape.
The ability of the prepared NPs to enhance paracellular transport was investigated in vitro in Caco-2 cell monolayers. The NPs with a positive surface charge (or shelled with CS) effectively reduced the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers. After removal of the incubated nanoparticles, a gradual increase in TEER was occurred. Confocal laser scanning microscopy observations verified that the NPs transiently opened the tight junctions between Caco-2 cells and facilitated transport of the NPs via the paracellular pathways. Furthermore, orally administered NPs were co-localized with the ZO-1 proteins at cell-cell contact sites in the small intestine of rats. This suggested that these NPs were able to interact and modulate the function of ZO-1 proteins, thus allowing transport of the NPs through the intestinal epithelium.
In the protein drug (insulin) loading process, increasing the amount of insulin used led to a larger size of the NPs together with a significant increase in their insulin loading efficiency and loading content. The insulin concentration used was 84.0 □g/ml, the loading efficiency and loading content of the NPs were approximately 55% and 15%, respectively. Insulin release profiles were significantly affected by their stability at distinct pH environments. No significant conformational change was observed for insulin released from the NPs at pH 7.4, as compared with that of the standard insulin. Moreover, in vivo results clearly indicated that oral administration of insulin in NPs in diabetic rats achieved a sustained effect in decreasing the blood glucose level over at least 10 h, suggesting that the effect of the proposed NPs enhanced absorption of fully functional insulin. These experimental results indicated that the prepared NPs increased absorption of insulin in the intestine and provided a protection from the GI environment. This novel NP system, which is composed of CS and γ-PGA, may be a suitable carrier for oral insulin delivery.

TABLE OF CONTENT
ABSTRACT……………………………………………………………….    I
TABLE OF CONTENT…………………………………………………..    III
LIST OF FIGURES AND TABLES…………………………………….. VI

Chapter 1.  Introduction    1

Chapter 2.    Preparation of Nanoparticles Composed of Chitosan/Poly-γ-Glutamic Acid and Evaluation of Their Permeability Through Caco-2 Cells
2-1 Materials and Methods    6
    2-1-1 Materials    6
    2-1-2 Depolymerization of CS by Enzymatic Hydrolysis    6
    2-1-3 Production and Purification of □-PGA    7
    2-1-4 Preparation of the CS–γ-PGA NPs    9
    2-1-5 Characterization of the CS–γ-PGA NPs    9
    2-1-6 Caco-2 Cell Cultures and TEER Measurements    10
    2-1-7 fCS–γ-PGA NP Preparation and CLSM Visualization    11
    2-1-8 Statistical Analysis    12
2-2 Results and Discussion    12
    2-2-1 Depolymerization of CS by Enzymatic Hydrolysis      12
    2-2-2 Production and Purification of □-PGA    14
    2-2-3 Preparation of the CS–γ-PGA NPs    15
    2-2-4 Characterization of the CS–γ-PGA NPs    17
    2-2-5 TEER Measurements    20
    2-2-6 CLSM Visualization    22
2-3 Conclusion    24

Chapter 3. Preparation and Characterization of Nanoparticles Shelled with Chitosan for Oral Insulin Delivery
3-1 Materials and Methods    26
3-1-1 Materials    26
    3-1-2 Preparation of Low MW CS and □-PGA    26
    3-1-3 Preparation of NPs    26
    3-1-4 Characterization of NPs    27
    3-1-5 TEER Measurements    27
    3-1-6 FITC-Labeled NPs and CLSM Visualization    28
    3-1-7 Visualization of Opening Tight Junctions    28
    3-1-8 Insulin Loaded Capacity of NPs    29
    3-1-9 In Vitro Release Study    30
    3-1-10 In Vivo Release Study    30
    3-1-11 Statistical Analysis    31
3-2 Results and Discussion    31
    3-2-1 Preparation of NPs    31
    3-2-2 Characterization of NPs at Distinct pH Values    34
    3-2-3 TEER Measurements    37
    3-2-4 CLSM Visualization    39
    3-2-5 Insulin Loaded Capacity of NPs    42
    3-2-6 In Vivo Release Study    43
3-3 Conclusion    45

Chapter 4. Novel Nanoparticles for Oral Insulin Delivery via The Paracellular Pathway for non-Invasive Diabetes Managements
4-1 Materials and Methods    47
4-1-1 Materials    47
    4-1-2 Preparation of Low MW CS and □-PGA    47
    4-1-3 Preparation of NPs    47
    4-1-4 Caco-2 Cell Cultures and TEER Measurements    48
    4-1-5 Fluorescence (FITC)-Labeled NPs and CLSM Visualization    48
    4-1-6 Stability and Functionality of NPs at Distinct pH Environments
    49
    4-1-7 Insulin-Loaded NPs    50
    4-1-8 Animal Study    51
    4-1-9 Statistical Analysis    52
4-2 Results and Discussion    52
    4-2-1 Preparation of NPs    53
    4-2-2 Stability of NPs at Distinct pH Environments    53
    4-2-3 Functionality of NPs at Distinct pH Environments    55
    4-2-4 Insulin-Loaded NPs    62
    4-2-5 Release of Insulin from Loaded NPs    65
    4-2-6 In Vivo Studies    66
4-3 Conclusion    67

References    68


著作目錄    77
作者簡歷    83

                                

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