多功能生物可分解奈米粒子做為生醫應用之研究

Multifunctional biodegradable nanoparticles for biomedical applications

李伯偉(Po-Wei Lee)

指導教授 ： 宋信文

國立清華大學/工學院/化學工程學系/博士(2009年)

摘要

As a highly immune-reactive tissue containing an abundance of antigen-presenting cells such as Langerhans cells, skin represents a favorable site for DNA immunization. Previous human clinical studies on the gene gun have demonstrated the feasibility of directly targeting LCs to deliver DNA-coated gold particles. However, when accumulated, gold particles as a carrier for transdermal gene delivery may incur adverse side effects. In the first part of this study, biodegradable nanoparticles, composed of chitosan (CS) and poly-γ-glutamic acid (γ-PGA), were prepared by an ionic-gelation method for transdermal DNA delivery (CS/γ-PGA/DNA) using a low-pressure gene gun. Conventional CS/DNA without the incorporation of γ-PGA were used as a control. The internal structures of test nanoparticles were then examined using small-angle X-ray scattering, while their constituents were identified using Fourier transformed infrared spectroscopy. CS/γ-PGA/DNA were spherical in shape with a relatively homogeneous size distribution. In contrast, CS/DNA had a heterogeneous size distribution with a donut, rod or pretzel shape. Both test nanoparticles could effectively retain the encapsulated DNA and protect it from nuclease degradation. Compared with CS/DNA, CS/γ-PGA/DNA enhanced their penetration depth into the mouse skin and enhanced the gene expression. Above observations may be attributed to that CS/γ-PGA/DNA were more compact in their internal structures and had a greater density than their CS/DNA counterparts, thus having a larger momentum to penetrate into the skin barrier. Experimental results indicated that CS/γ-PGA/DNA may substitute gold particles as a DNA carrier for transdermal gene delivery. In the second part of this study, a multifunctional core-shell nanoparticle system was developed, which can be delivered transdermally into the epidermis by a gene gun as a DNA carrier. The developed nanoparticles were consisted a hydrophobic PLGA core and a positively-charged glycol chitosan (GC) shell. Based on use of the core of nanoparticles, fluorescent quantum dots (QDs) were loaded for ultrasensitive detection of the migration of Langerhans cells once delivered transdermally, while a reporter gene was electrostatically adsorbed onto the GC shell layer of nanoparticles. Results obtained from fluorescence spectrophotometry, transmission electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction measurement indicated that the prepared nanoparticles had a core-shell structure with QDs in their core area. The surface charge of nanoparticles was strongly dependent on their pH environments, allowing the release of the loaded DNA intracellularly through a pH-mediated mechanism. Based on use of a mouse model, our results further demonstrated that bombardment of nanoparticles transfected DNA directly into LCs present in the epidermis. The transfected LCs then migrated and expressed the encoded gene products in the draining lymph nodes. Above results suggest the feasibility of using the developed nanoparticle system to monitor and fine-tune important functional aspects of the immune system, in conjunction with the loaded fluorescence, thus having the potential for use in immunotherapy and vaccine development. This study also explored the feasibility of using the CSNP system to develop a MRI constrast. Results of this study demonstrate that an efficient contrast agent for magnetic resonance imaging (MRI) is essential to enhance the detection and characterization of lesions within the body.
This study described the feasibility of developing biodegradable nanoparticles with a core-shell structure to formulate superparamagnetic iron oxide (CSNP-SPIO) for MRI. The developed nanoparticles were composed of a hydrophobic PLGA core and a positively-charged glycol chitosan shell. Results obtained from transmission electron microscopy, energy dispersive X-ray analysis, electron energy loss spectroscopy, and X-ray diffraction measurement indicated that the prepared nanoparticles had a core-shell structure with SPIO in their core area. Nanoparticles did not aggregate together during storage in water, owing to the electrostatic repulsion between positively-charged nanoparticles. The magnetic properties of nanoparticles were then examined by a vibrating sample magnetometer and a superconducting quantum interference device. Experimental results indicated that the superparamagnetism of SPIO was preserved after the CSNP-SPIO formulation. Closely examining their cellular internalization pathway revealed that CSNP-SPIO accumulated in lysosomes. In the biodistribution study, a high level of radioactivity was observed in the liver shortly after administering the 99mTc-labeled CSNP-SPIO intravenously. Once taken up by the liver cells, the liver turned dark on T2* images. Following cellular internalization, CSNP-SPIO were broken down gradually. Therefore, as time increased, the darkness of the liver on T2* images significantly decreased. Results of this study demonstrated the developed CSNP-SPIO can serve as an efficient MRI contrast agent and could be degraded after serving in their imaging function.

關鍵字

穿皮基因傳遞 ； 生物可分解奈米微粒 ； 基因槍 ； 細胞追蹤 ； 免疫治療 ； 酸鹼敏感性材料 ； 胞飲路徑

並列摘要

無資料

並列關鍵字

transdermal gene delivery ； degradable nanoparticle ； gene gun ； quantum dots ； cell tracking ； immunotherapy ； stimuli-responsive materials ； superparamagnetic iron oxide ； multifunction ； internalization pathway

參考文獻

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