利用聚麩胺酸-聚乳酸團聯共聚物製備奈米微粒以標的肝癌細胞之研究：體外及體內實驗

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研究生：	梁祥發 Hsiang-Fa Liang
論文名稱：	利用聚麩胺酸-聚乳酸團聯共聚物製備奈米微粒以標的肝癌細胞之研究：體外及體內實驗 Nanoparticles composed of poly(gamma-glutamic acid)-poly(lactide) block copolymers for targeting hepatoma cells: In vitro and in vivo studies
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：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	103
中文關鍵詞：	奈米微粒、藥物制放系統、主動標的、抗癌藥物
外文關鍵詞：	Nanoparticle, Drug delivery system, Active targeting, Anti-tumor efficacy
相關次數：	點閱：2 下載：0
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In the study, poly(□-glutamic acid) (□-PGA) and poly(lactide) (PLA) were used to synthesize block copolymers via a simple coupling reaction between □-PGA and PLA to prepare self-assembled nanoparticles (NPs). For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (Gal-NPs) as a targeting moiety.
The prepared NPs had a mean particle size of about 140 nm with a zeta potential of about -20 mV. The results obtained by the TEM and AFM examinations showed that the morphology of the prepared NPs was spherical in shape with a smooth surface. Additionally, it was found that no aggregation or precipitation of NPs was observed during storage for up to 1 month, as a result of the electrostatic repulsion between the negatively charged NPs. Moreover, in the cellular uptake study, it was found that the Gal-NPs prepared in the study had a specific interaction with HepG2 cells via ligand-receptor recognition.
In the paclitaxel encapsulation study, with an increase of the paclitaxel/polymer ratio form 0.5/10 to 3/10, the mean particle size of the prepared NPs increased from 115 to 260 nm, while the loading efficiency decreased from 76% to 21%. In the in vitro cell viability study, the dose dependent tendency of cytotoxicity for the paclitaxel-loaded Gal-NPs was comparable with that of Taxol□ formulation, and was significantly greater than that of the paclitaxel-loaded NPs. It was found by the immunocytochemistry and flow cytometric studies that the paclitaxel released from the prepared NPs or Gal-NPs could arrest cellular microtubules and altered HepG2 cells in specific cell cycle stages.
The biodistribution study in normal or hepatoma tumor-bearing nude mice showed that the Gal-NPs were accumulated mainly in the liver. In contrast, the NPs were distributed mainly in spleen. Additionally, the cumulative amount of the Gal-NPs in the tumor site was considerable greater than that of the NPs.
It was noted in the in vivo anti-tumor study that only the paclitaxel-loaded Gal-NPs could significantly reduce the tumor volume and inhibited the tumor growth. Furthermore, the body weight of the paclitaxel-loaded Gal-NPs treated mice were maintained constant. The results indicated that the paclitaxel-loaded Gal-NPs were mainly accumulated in the tumor site due to a higher permeability of tumor vessels and ligand-receptor recognition by hepatoma cells.
The aforementioned results indicated that the prepared nanoparticles composed of γ-PGA-PLA block copolymers conjugated with galactosamine may be a potential drug delivery system for targeted delivery to liver cancers or other liver diseases.

Table of Content
ABSTRACT    I
TABLE OF CONTENT    III
LIST OF FIGURES AND TABLES    VIII

Chapter 1.  Introduction    1
Chapter 2.      Preparation of nanoparticles composed of poly(γ-glutamic acid)-poly(lactide) block copolymers and evaluation of their uptake by HepG2 cells

1 Materials and Methods    6
1-1 Materials    6
1-2 Production and purification of □-PGA    7
1-3 Hydrolysis and analysis of □-PGA    9
1-4 Synthesis of γ-PGA-PLA block copolymers    10
1-5 Nanoparticle preparation and characterization    11
1-6 Fluorescence spectroscopy (pyrene)    12
1-7 Conjugation of galactosamine to the surface of nanoparticles    13
1-8 Cell uptake study    13
1-9 Cytotoxicity of nanoparticles    14
1-10 Statistical analysis    15
2 Results and Discussion    15
2-1 Production and purification of □-PGA    16
2-2 Hydrolysis of □-PGA    18
2-3 Synthesis of γ-PGA-PLA block copolymers    19
2-4 Characteristics of nanoparticles    21
2-5 Critical micelle concentration (CMC)    24
2-6 Cellular uptake of the prepared nanoparticles    26
3 Conclusion    30

Chapter 3.  Paclitaxel-loaded poly(γ-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells

1 Materials and Methods    31
1-1 Materials    31
1-2 Production of □-PGA    32
1-3 Hydrolysis of □-PGA    32
1-4 Synthesis of γ-PGA-PLA Block Copolymers    34
1-5 Preparation of the Paclitaxel-loaded Nanoparticles    35
1-6 Loading Content and Loading Efficiency of the Paclitaxel-loaded Nanoparticles    36
1-7 Release of Paclitaxel from the Loaded Nanoparticles    37
1-8 Conjugation of Galactosamine to the Paclitaxel-loaded Nanoparticles    37
1-9 Viability of HepG2 Cells Treated with Distinct Paclitaxel Formulations    38
1-10 Immunofluorescence Analysis of HepG2 Cells Treated with Distinct Paclitaxel Formulations    39
1-11 Altered Cycling States of HepG2 Cells Treated with Distinct Paclitaxel Formulations    40
1-12 Statistical Analysis    40
2 Results and Discussions    41
2-1 Production of □-PGA    41
2-2 Hydrolysis of □-PGA    41
2-3 Synthesis of γ-PGA-PLA block copolymers    43
2-4 Particle Size and Morphology of the Paclitaxel-loaded Nanoparticles    44
2-5 Loading Content and Loading Efficiency of the Paclitaxel-loaded Nanoparticles    48
2-6 Release of Paclitaxel from the Loaded Nanoparticles    49
2-7 Conjugation of Galactosamine to the Paclitaxel-loaded Nanoparticles    50
2-8 Viability of HepG2 Cells Treated with Distinct Paclitaxel Formulations    51
3 Conclusion    58


Chapter 4. Biodistribution and anti-tumor efficacy of paclitaxel-loaded □-PGA-PLA nanoparticles as ASGP receptor targeted drug delivery system
1 Materials and Methods    59
1-1 Materials    59
1-2 Synthesis of γ-PGA-PLA block copolymers    60
1-3 Preparation of paclitaxel-loaded nanoparticles    61
1-4 Conjugation of galactosamine to the paclitaxel-loaded nanoparticles    62
1-5 Loading content and loading efficiency of the prepared nanoparticles    63
1-6 Release profiles of paclitaxel from the prepared nanoparticles    63
1-7 Viability of HepG2 cells treated with distinct paclitaxel formulation    64
1-8 Immunofluorescence analysis of HepG2 cells treated with distinct paclitaxel formulations    65
1-9 Studied animals    66
1-10 Biodistribution of the prepared nanoparticles    66
1-11 Anti-tumor efficacy of the prepared nanoparticles    67
1-12 Statistical analysis    67
2 Results and Discussions    68
2-1 Synthesis of □-PGA-PLA block copolymers    68
2-2 Characteristics of the paclitaxel-loaded nanoparticles    69
2-3 Release of paclitaxel from the prepared nanoparticles    71
2-4 Viability of HepG2 cells treated with distinct paclitaxel formulations    73
2-5 Biodistributions of the prepared nanoparticles in normal and nude mice    77
2-5 Anti-tumor efficacy of the prepared nanoparticles    82
3 Conclusion    84

Chapter 5.  Summary    85

References    87

著作目錄    96

個人獎譽    102

作者簡歷    103

                                

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