Novel pH-Sensitive Chitosan Nanoparticles for Oral Insulin Delivery: Mechanism, Biodistribution, Toxicological, Pharmacodynamic and Pharmacokinetic Evaluations

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研究生：	桑傑 Kiran Sonaje
論文名稱：	Novel pH-Sensitive Chitosan Nanoparticles for Oral Insulin Delivery: Mechanism, Biodistribution, Toxicological, Pharmacodynamic and Pharmacokinetic Evaluations 新穎具pH敏感性幾丁聚醣奈米微粒口服胰島素載體之機制,生物分佈,毒理學,藥物動力學及效學探討
指導教授：	宋信文 Sung, Hsing-Wen
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：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	97
中文關鍵詞：	幾丁聚醣奈米微粒、胰島素、口服釋放、機制、生物分布、安全性
外文關鍵詞：	Chitosan nanoparticles, Insulin, Oral Delivery, Mechanism, Biodistribution and Pharmacokinetics, Safety
相關次數：	點閱：2 下載：0
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The present dissertation was aimed at evaluating the mechanism, safety and efficacy of novel pH-sensitive nanoparticles (NPs) for the oral delivery of insulin. The self-assembled NPs with pH-responsive characteristics were prepared by mixing the anionic poly-r-glutamic acid solution with the cationic chitosan (CS) solution in the presence of MgSO4 and sodium tripolyphosphate. The absorption enhancing potential of the test NPs was evaluated using an in vitro Caco-2 cell monolayer model. The in vitro results indicated that the transport of insulin across the cell monolayers was improved by NPs in a pH-dependent manner; with an increase in pH, the amount of insulin transported decreased significantly. The impact of orally administered NPs on the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin was evaluated in a diabetic rat model. Oral administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at least 10 h in diabetic rats and the corresponding relative bioavailability of insulin was found to be 15.1□ 0.9%.
The test NPs were then used to deliver the aspart-insulin, a monomeric insulin analogue. The biodistribution of orally administered aspart-insulin via NPs was studied in rats using the single-photon emission computed tomography (SPECT)/computed tomography (CT). The results indicated that the insulin was absorbed into the systemic circulation, while the drug carrier (CS) was mainly retained in the gastrointestinal tract. The biodistribution of orally administered aspart-insulin was then compared that administered via subcutaneous (SC) injection. In the PD/PK evaluation in a diabetic rat model, the orally administered aspart-insulin-loaded NPs produced a slower hypoglycemic response for a prolonged period of time, whereas the SC injection of aspart-insulin produced a more pronounced hypoglycemic effect for a relatively shorter duration. Finally, comparison of the PD/PK profiles of the orally administered aspart-insulin with those of the SC injection of NPH-insulin (an intermediate-acting insulin preparation) suggested the suitability of our NP system as a noninvasive alternative for the basal insulin therapy.
It has been suggested that the paracellular permeation enhancement by CS may result in the absorption of unwanted toxins present in the GI tract. Therefore, the effects of test NPs on the oral absorption of lipopolysaccharide (LPS, a model toxin) were evaluated in animal models. The results indicated that the intestinal mucus layer acts as a barrier for the absorption of orally administered LPS. However, the test NPs could infiltrate the mucus layer due to their positive surfaces and were able to enhance the absorption of loaded insulin alone without affecting the absorption of LPS. Additionally, the toxicity study confirmed that test NPs did not improve the oral absorption of LPS.
Finally, the ultrastructural effects of CS on the epithelial tight junctions and paracellular permeability were investigated using transmission electron microscopy (TEM). The results indicated that the interaction of CS with intestinal cells induced a relatively rapid increase in paracellular permeability both in vitro and in vivo. The mechanism of action of CS with regard to its ability to disrupt epithelial cell TJs was found to be due to the translocation of the TJs proteins ZO-1, occludin and JAM-1 from the plasma membrane into the cytosol. The TEM results revealed no detectable alterations in the morphology of the TJs or lateral intercellular spaces, indicating that the TJ opening activity of CS was transient and reversible. These results indicated that the prepared NPs can serve as a safe and effective alternative for the oral delivery of insulin

Abstract..…………………………………………………………….…….i
Table of contents    iii
List of Figures    vii
List of Tables    x
Chapter 1.     Introduction    ………………………………………...1
Chapter 2.    In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery    5
1. Materials and Methods    5
1.1. Preparation and characterization of test NPs    5
1.2. Fluorescent NP preparation and confocal laser scanning microscopy (CLSM) visualization    7
1.3. Insulin transport study    7
1.4. Animal studies    8
1.5. In vivo mucoadhesion and intestinal permeation of NPs    8
1.6. In vivo toxicity study    9
1.7. Pharmacological activity of the insulin loaded in NPs    9
1.8. Oral efficacy and bioavailability of NPs    10
1.9. Statistical analysis    11
2. Results    11
2.1. Characterization of insulin-loaded NPs    11
2.2. CLSM visualization of Caco2 monolayers    12
2.3. Insulin transport study    15
2.4. In vivo mucoadhesion and intestinal permeation of NPs    15
2.5. In vivo toxicity    16
2.6. Pharmacological activity of the insulin loaded in NPs    19
2.7. Oral efficacy and bioavailability    20
3. Discussion    22
4. Conclusions    26
Chapter 3.     Biodistribution, Pharmacodynamics and Pharmacokinetics of Insulin Analogues in a Rat Model: Oral Delivery Using pH-Responsive Nanoparticles vs. Subcutaneous Injection    27
1. Materials and Methods    29
1.1. Preparation of the insulin-loaded NPs    29
1.2. Characterization of test NPs    30
1.3. In vitro Caco-2 cell monolayer experiments    30
1.4. Animal studies    32
1.5. Biodistribution study    32
1.6. PD/PK study    34
1.7. Statistical analysis    34
2. Results and Discussion    35
2.1. Characterization of the aspart-insulin loaded NPs    36
2.2. TEER measurement, drug transport and CLSM visualization in Caco-2 cell monolayers    38
2.3. Biodistribution study    41
2.4. PD/PK study    45
3. Conclusions    47
Chapter 4.    Effects of Chitosan Nanoparticles on the Oral Absorption of Unwanted Toxins Present in the Intestinal Lumen: An In vivo Evaluation Using Bacterial Lipopolysaccharide as a Model Toxin    48
1. Materials and Methods    50
1.1.    Preparation and characterization of test NPs    50
1.2.    Evaluation of the micelle forming characteristics of LPS    50
1.3.    Animal studies    51
1.4.    Biodistribution study    51
1.5.    Ultrastructural examination of the in vivo tight junction opening activity of CS-NPs    52
1.6.    GI absorption of FITC-LPS and Cy3-insulin-loaded-NPs    53
1.7. In vivo toxicity study    54
1.8. Statistical analysis    55
2. Results and discussions    55
2.1. Characterization of the insulin-loaded NPs    56
2.2. Oral absorption and biodistribution of LPS    56
2.3. Effects of CS-NPs on the absorption and biodistribution of LPS and insulin    58
2.4. Micelle forming characteristics of LPS    60
2.5. Ultrastructural examination of TJ opening activity of CS in vivo    62
2.6. In vivo CLSM study    64
2.7. In vivo toxicity study    67
3. Conclusions    69
Chapter 5.    Effects of chitosan on the epithelial TJs and paracellular permeability: An ultra-structural examination….…………70
1. Materials and Methods    72
1.1. Synthesis of Fluorescein isothiocyanate-labeled CS    72
1.2. Synthesis of Quantum Dots-labeled CS    72
1.3. Synthesis of Cy3-labeled Insulin    72
1.4. TEER measurements and in vitro CLSM visualization    73
1.5. Ultrastructural examination of TJ opening activity of CS in vitro    74
1.6. Animal studies    75
1.7. GI absorption of fluorescent-NPs studied using CLSM    75
1.8. Ultrastructural examination of TJ opening activity of CS in vivo    76
2. Results and discussions    76
2.1. TEER measurements    77
2.2. CLSM visualization of Caco-2 cell monolayers    78
2.3. Ultrastructural examination of TJ opening activity of CS in vitro    80
2.4. In vivo absorption of fluorescent NPs    81
2.5. Ultrastructural examination of TJ opening activity of QD-CS in vivo    82
4. Conclusions    85
References….    86
List of Publications    95
Biography……    97

                                

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