臺灣博碩士論文加值系統

非病毒式載體（non-viral vectors）相較於病毒式基因載體（viral vectors）目前已被認為是較具生物安全性的基因載體。在眾多製作基因載體的材料中，聚乙烯亞胺（PEI）因對pH值的變化具有緩衝能力（pH-buffering），可保護基因不會因酸化而被分解，並可促進內質體破裂而釋放出所包覆的基因，是相當有潛力的材料之一。其中分子量越高(>25kDa)的聚乙烯亞胺（polyethyleneimine, PEI）可提供較高的基因轉殖效率，但相對也具有較高的生物毒性，使得聚乙烯亞胺在臨床應用上的發展受到限制。

由於先前的實驗已證實經由硬脂酸修飾聚乙烯亞胺可降低其生物毒性，並提高低分子量聚乙烯亞胺的轉殖效率，以硬脂酸修飾後的聚乙烯亞胺為帶有正電荷之雙極性聚合物，可進一步製成奈米載體（PEI-SA），同時傳輸帶負電荷之siRNA與疏水性抗癌藥物doxorubicin，然而此載體在藥物釋放時仍有所限制，有待進一步克服此瓶頸。因此，本研究以不同重量比例混合之10k PEI與 1.8k PEI製成載體，除了可維持其原有攜帶基因與藥物之功能及低生物毒性等特性外，亦可藉由低分子量PEI的加入以調整其結構，增加其藥物釋放的效率。此外，PEI-SA亦可包覆超順磁氧化鐵（SPIO）作為顯影劑應用於活體影像之攝影。相較於市售之影像對比劑Resovist，經由PEG修飾之PEI-SA包覆SPIO（PEI-SA/SPIO）在注入BALB/c老鼠後的實驗結果可證實其在血液中有較長的半衰期，且顯影之效果與市售產品Resovist相當。

銀耳多醣目前已被廣泛應用於食品、中草藥及疫苗佐劑，已有許多文獻證實其具有抗癌及抗發炎之反應，為增加奈米顆粒之生物應用性，本研究同時將銀耳多醣修飾製成帶有正電荷之聚合物，再以硬脂胺接枝於其上進行改質，以溶劑發散法（oil-in-water solvent evaporation method）將其製成表面帶正電的聚合多醣奈米顆粒之後，以動態光散射法（dynamic light scattering）測得其大小為129–421nm，水中表面電位為63–73 mV ，另以AFM、TEM及FTIR測定其物理特性，此外，亦測定其攜帶基因的能力、細胞攝取效率（cellular uptake efficiency）及細胞毒性。最後將此銀耳多醣製成之奈米顆粒加入LPS活化後的巨噬細胞，測量其一氧化氮之生產量以評估其抗發炎反應，結果顯示其具有抑制發炎反應生成之能力。藉由銀耳多醣奈米顆粒可攜帶基因、疏水性分子及顯影劑SPIO之能力，同時亦具有抑制發炎反應生成之特性，可預期此奈米顆粒對於慢性發炎組織之標靶藥物傳輸及相關影像造影的應用將具有相當之潛力。

Non-viral gene carriers composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation, and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer such that high molecular weight (>25kDa) of PEI can elevate the transfection efficiency. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, and improve the insufficient transfection efficiency of lower molecular weight PEI.
Cationic amphiphilic copolymer consisted of stearyl side chains on polyethyleneimine (PEI) main chain (PEI-SA) was developed previously and demonstrated with the concept of co-delivering siRNA and anti-tumor drug doxorubicin. However, the drug release profile was limited and remained to be an issue to be overcome. In the present study, hybrid PEI in different weight ratios of 10k: 1.8k was proposed to alter this structural formulation by incorporating with low molecular weight PEI. The design was able to maintain the functionalities as gene and drug carrier with efficient binding capability, enhanced drug release rate, also optimized between cellular uptake and low cellular cytotoxicity. Other functionality was also attempted to integrate into the PEI-SA nanoparticles by encapsulation with the SPIOs to formulate as contrast agents for in vivo imaging application. BALB/c mice was injected with PEG conjugated PEI-SA/SPIO nanoparticles to demonstrate the extended half-life in blood plasma, and effective contrast agents comparable to the commercial available contrast agents Resovist.
A new type of polymeric polysaccharide nanoparticles was also proposed and developed. Tremella polysaccharides have been commonly used as herbal medicine, vaccine adjuvant, or orally fed for anti-tumor or anti inflammatory studies. To date, none of them has been formulated as nanoparticles and applied for biological studies. The fruit body of Tremella fuciformis was extracted and cationic modified, followed by oil-in-water solvent evaporation method to formulate into nanoparticles. The physical characteristics of these nanoparticles were then confirmed by dynamic light scattering, AFM, TEM and FTIR with size of 107.1±2.5 nm and zeta potential of 70.6±3.3mV. The tremella nanoparticles were found with enhanced cellular uptake and relatively low cytotoxicity. Gene binding capacity was also investigated to ensure the functionality as potential gene carriers. The anti-inflammatory capability was demonstrated by measuring the nitric oxide produced from LPS-activated macrophages. The use of nano-sized tremella polysaccharide nanoparticles can posses opportunities as delivery carriers for gene and contrast agent by incorporating hydrophobic SPIO to target macrophage-rich tissue at chronic inflammation site.

摘要 i
Abstract iii
Table of Content vi
List of Figures ix
List of Tables xi
List of Equations xii
CHAPTER 1 INTRODUCTION 1
1.1 Nanotechnology 1
1.1.1 Contrast Agents 1
1.1.2 Gene delivery 5
1.2 Polymeric nanoparticles 6
1.3 Polysaccharide Nanoparticles 9
1.3.1 Chitosan 12
1.3.2 Tremella 14
1.4 Inflammation 16
1.4.1 Nitric Oxide 17
1.4.2 Macrophages 19
1.5 Aim 22
CHAPTER 2 MATERIAL AND METHODS 25
2.1 Synthesis of PEI-SA nanoparticles 25
2.1.1 PEI-SA 25
2.1.2 Preparation and characterization of PEG conjugated PEI-SA/SPIO nanoparticles 26
2.1.3 in vivo MRI imaging 27
2.2 Tremella 27
2.2.1 Tremella polysaccharide 27
2.2.2 Preparation of cationic tremella polysaccharide 28
2.2.3 Hydrolysis for tremella polysaccharide 29
2.2.4 Formulation of tremella nanoparticles 30
2.3 Characterization of nanoparticles 30
2.3.1 Particle size and zeta potential 30
2.3.2 Atomic force microscopy and transmission electron microscopy 31
2.3.3 Encapsulation and release of doxorubicin in PEI-SA nanoparticles 31
2.3.4 Fourier transform infrared spectroscopy 32
2.4 Gel retardation 32
2.5 Cytotoxicity study 33
2.6 siRNA cellular uptake evaluation 34
2.6.1 Flow cytometry 34
2.7 Anti-inflammatory evaluation 35
2.7.1 Nitric oxide evaluation 35
CHAPTER 3 RESULTS 36
3.1 Hybrid 10k:1.8k PEI-SA nanoparticles 36
3.1.1 Characteristics of hybrid PEI-SA nanoparticles in various weight ratios of 10k:1.8k PEI polymer 36
3.1.2 Binding efficiency 38
3.1.3 Cytotoxicity analysis 40
3.1.4 Assesment of cellular uptake 45
3.1.5 Drug release profile 47
3.2 Characterization of PEI-SA/SPIO nanoparticles 49
3.2.1 Encapsulation of SPIO in 10k PEI-SA nanoparticles 49
3.2.2 Encapsulation and loading efficiency of SPIO in PEI-SA nanoparticles 51
3.2.3 in vivo MRI imaging of Balb/c mice injected with contrast agents 53
3.2.4 Quantitative study of MRI signal intensity in liver, kidney, prostate, and brain 55
3.3 Characterization of tremella polysaccharide nanoparticles 57
3.3.1 SEM images of native tremella polysaccharide 57
3.3.2 Cationic modification of native tremella polysaccharide 59
3.3.3 Physical characteristics of tremella nanoparticles 61
3.3.4 TEM and AFM images of tremella nanoparticles 63
3.3.5 FTIR spectrum 66
3.3.6 Binding efficiency 68
3.3.7 Cytotoxicity analysis 70
3.3.8 Cytotoxicity analysis – cell type study 72
3.3.9 Cellular uptake of tremella nanoparticles copmlex 74
3.3.10 Anti-inflammatory 76
3.3.10.1 Macrophages treated with lipopolysaccharides and tremella nanoparticles 76
3.3.10.2 Macrophages treated with lipopolysaccharides followed by tremella nanoparticles 78
CHAPTER 4 DISCUSSION 80
CHAPTER 5 CONCLUSION 93
REFERENCE 95

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