||The development of multifunctional nanocomplexes for drug delivery and oral cancer therapy
||Institute of Oral Medicine
superparamagnetic iron oxide nanoparticles
drug delivery system
口腔癌在台灣的發生率逐漸上升，同時在十大癌症死亡率排名第五位。有鑑於此，針對口腔癌發展出有效的治療方法便成為一個重要的課題。本研究主要是利用超順磁性氧化鐵奈米粒子結合海藻膠來開發出一個可應用於口腔癌治療的藥物載體。海藻膠與超順磁性氧化鐵奈米粒子結合所形成之奈米複合材料(Fe3O4/alginate nanoparticles)的製備方式是先混合氨基型四氧化三鐵奈米粒子以及海藻膠，再以碳二亞胺反應幫助胜肽鍵形成。Fe3O4/alginate nanoparticles的水合直徑、表面電位和表面修飾則分別以動態光散射儀和傅立葉轉換紅外線光譜進行分析。Doxorubicin (DOX)的裝載是將DOX與Fe3O4/alginate nanoparticles先行混合後，再將混合液加入氯化鈣溶液中，形成有裝載DOX的Fe3O4@Ca-alginate nanoparticles。藥物包裹率以及裝載量的計算是以螢光測定結果計算而得；而藥物的釋放率則是將裝載DOX的Fe3O4@Ca-alginate nanoparticles置於不同的溶液環境中，每24小時監測一次，連續監測7天。結果顯示Fe3O4/alginate nanoparticles的水合直徑為76.5±19.2奈米，表面電位為-39.0±0.3毫伏特，從傅立葉轉換紅外線光譜的結果亦證實了海藻膠成功結合到四氧化三鐵奈米粒子的表面。當DOX與鐵的重量比為8:1時，藥物的包裹率和裝載量會達到最高。藥物釋放曲線顯示裝載DOX的Fe3O4@Ca-alginate nanoparticles在細胞質模擬緩衝溶液的環境中有最高的釋放率。細胞毒性測試結果顯示裝載DOX的Fe3O4@Ca-alginate nanoparticles其細胞毒性與純藥物相當。綜合上述的結果，Fe3O4@Ca-alginate nanoparticles可做為藥物輸送系統，期望在未來可以利用這個系統提高腫瘤治療的效率，同時可降低藥物的副作用。
The incidence of oral cancer increases gradually and oral cancer is the fifth leading cause of cancer mortality in Taiwan. Thus, it is important to develop an effectively therapeutic approach for oral cancer. The aim of this study is to develop a drug delivery system made of superparamagnetic iron oxide nanoparticles (SPIONs) covalently linked with alginate for oral cancer therapy. Alginate-conjugated SPIONs (Fe3O4/alginate nanoparticles) is prepared by mixing NH3+-exposed magnetite (Fe3O4) nanoparticles and alginate, followed by carbodiimide reaction. The hydrodynamic diameter, zeta potential and surface modification of Fe3O4/alginate nanoparticles are characterized by dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FT-IR). Doxorubicin (DOX) is encapsulated into the drug delivery system by mixing DOX and Fe3O4/alginate nanoparticles solution followed by addition of the mixture into CaCl2 aqueous solution (DOX-loaded Fe3O4@Ca-alginate nanoparticles). The encapsulation efficiency and loading capacity are determined by fluorescence spectrometer with excitation wavelength of 485 nm and emission wavelength of 590 nm; release profiles of DOX-loaded Fe3O4@Ca-alginate nanoparticles in different medium are monitored from 24 hr to 7 days. The hydrodynamic diameter and zeta potential of Fe3O4/alginate nanoparticles are 76.5±19.2 nm and -39.0±0.3 mV, respectively, and the FT-IR spectra of SPION-alginate reveals that alginates conjugate successfully to Fe3O4 nanoparticles. The encapsulation efficiency and loading capacity exhibit maximum values when the weight ratio of DOX to Fe is 8:1. The release profile indicates that the cumulative DOX release percentage is highest at the cytoplasm mimicking buffer. The in vitro cytotoxicity of DOX-loaded Fe3O4@Ca-alginate nanoparticles shows similar degree of cytotoxicity to that of free DOX. Our results suggest that Fe3O4@Ca-alginate nanoparticles can serve as a drug nanocarrier, and this system has a great potential to improve therapeutic efficiency and minimize side effects of anticancer drugs in the future.
Figure Contents VIII
1. Introduction 1
1-1 Drug delivery system 1
1-2 Magnetic nanoparticle 2
1-3 Magnetic behaviors of SPION 3
1-4 Synthesis of SPIONs 5
1-5 SPIONs coated by alginate 7
1-6 Alginate 8
1-7 Preparation of alginate hydrogel 9
1-7.1 Ionotropic gelation of alginate 9
1-7.2 Alginate acid gel 10
1-8 Study purpose 10
2. Materials and Methods 12
2-1 Materials 12
2-1.1 Synthesis of Fe3O4 and Fe3O4/alginate nanoparticles 12
2-1.2 Cell culture 12
2-1.3 Drug release experiment 13
2-1.4 MTT assay 13
2-2. Methods 13
2-2.1 Preparation of Fe3O4 nanoparticles 13
2-2.2 Preparation of Fe3O4/alginate nanoparticles 14
2-2.3 Ionic crosslinking of Fe3O4/alginate nanoparticles 14
2-2.4 Morphology and zeta potential of Fe3O4 nanoparticles 15
2-2.5 Morphology, hydrodynamic diameter and zeta potential of Fe3O4/alginate nanoparticles and Fe3O4@Ca-alginate nanoparticles 15
2-2.6 Fourier transform infrared spectroscopy (FT-IR) analysis of Fe3O4 nanoparticles, alginate and Fe3O4/alginate nanoparticles 16
2-2.7 Cell culture of human oral squamous cell carcinoma (OSCC) cell line, normal cell line and control normal keratinocyte 16
2-2.8 In vitro cytotoxicity studies of Fe3O4/alginate nanoparticles and Fe3O4@Ca-alginate nanoparticles 17
2-2.9 Preparation of DOX-loaded Fe3O4@Ca-alginate nanoparticles 17
2-2.10 Doxorubicin loading content and encapsulation efficiency 17
2-2.11 In vitro release profile of DOX-loaded Fe3O4@Ca-alginate nanoparticles 18
2-2.12 In vitro cytotoxicity evaluation of DOX-loaded Fe3O4@Ca-alginate nanoparticles 18
3. Results 19
3-1 Characterization of Fe3O4 nanoparticles 19
3-2 Characterization of Fe3O4/alginate nanoparticles and Fe3O4@Ca-alginate nanoparticles 19
3-3 FT-IR analysis of Fe3O4 nanoparticles, alginates and Fe3O4/alginate nanoparticles 20
3-4 In vitro cytotoxicity evaluation of Fe3O4/alginate nanoparticles and Fe3O4@Ca-alginate nanoparticles 20
3-5 Doxorubicin loading content and encapsulation efficiency 20
3-6 In vitro release profiles of DOX-loaded Fe3O4@Ca-alginate nanoparticles 21
3-7 In vitro cytotoxicity evaluation of DOX-loaded Fe3O4@Ca-alginate nanoparticles 21
4. Discussion 23
5. Conclusion 31
6. References 32
7. Figures and Legends 38
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