合成奈米材料及其在生醫與能源上之應用

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(1)國立臺灣師範大學化學系. 博士論文. 合成奈米材料及其在生醫與能源上之應用 Synthesis of Nanomaterials for Biomedical and Energy Applications. 李政宏 Li Cheng-Hung. 指導教授: 陳家俊博士 Advisor: Chia-Chun Chen, Ph. D. 中華民國 104 年 11 月 November, 2015.

(2) Abstract Recent development of molecular imaging probes for fluorescence-guided surgery has shown great progresses for precisely determining tumor margin to execute the tissue resection. Here we synthesize the fluorescent nanoparticles (gold and europium-doped gadolinium oxide) conjugated with nucleolin-targeted AS1411 aptamer.. The. nanoparticle. conjugates. exhibit. high. water-solubility,. good. biocompatibility, visible fluorescence, strong X-ray attenuation for computed tomography(CT). contrast. enhancement. and. high. magnetic. susceptibility. (europium-doped gadolinium oxide (Gd2O3:Eu) nanoparticles) . The fluorescent nanoparticle conjugates are applied as a molecular contrast agent to reveal the tumor location in CL1-5 tumor-bearing mice by CT imaging. Furthermore, the fluorescence emitting from the conjugates in the CL1-5 tumor can be easily visualized by the naked eyes. After the resection, the IVIS measurements show that the fluorescence signal of the nanoparticle conjugates in the tumor is greatly enhanced in comparison to that in the controlled experiment. The fluorescent imaging clearly reveals that the nanoparticles can be applied as fluorescent tags for cancer-targeting molecular imaging. Our work has shown potential application of functionalized nanoparticles as a multi-function imaging agent in clinical fluorescence-guided surgery. Overall, our results demonstrate that the fluorescence nanoparticles could not only be served as new medical contrast agents but also as intraoperative fluorescent imaging probes for guided surgery in the near future. Nanomaterials not only use on bio-application but also energy storage source, such as lithium battery. For more than a decade, scientists have tried to improve lithium-based batteries by replacing the graphite in one terminal with silicon, which can store 10 times more charge. But after just a few charge / discharge cycles, the. I.

(3) silicon structure would crack and crumble, rendering the battery useless. The new silicon based anodic materials in lithium ion battery (Si-based LIB) are worldwide developed to overcome the capacity decay during the lithiation/delithiation process. In this study, Si nanoparticles coated with 5-sulfoisophthalic acid (SPA) doped polyaniline (core/shell SiNPs@PANi/SPA) composite were prepared and applied as the anodic materials for LIB applications. The detailed structure of core/shell SiNPs@PANi/SPA composite was characterized by high-resolution scanning electron microscopy before and after the charging/discharging. The electrochemical measurements showed that the SiNPs@PANi/SPA anode exhibited high capacity of 925 mAh g-1 and high Columbic efficiency (99.6%) after long-term cyclic life (1000 cycles). Overall results indicated that the SPA dopant doped polyaniline served as a conductive matrix to improve electrical contact and to provide adhesive force in Si-based LIB. Our approach opens a route for the design of efficient silicon nanocomposites for LIB applications. Not only one way we want to approach high performance on anode of battery. We tried different materials like carbon-based metal oxide. Nanostructure composites of lead oxide/copper–carbon (PbO/Cu–C) were synthesized through in situ solvothermal synthesis and heat treatment of PbO/Cu with polyvinylpyrrolidone (PVP) and used as lithium-ion battery anodes. A PbO active particle was embedded in the Cu and PVP–C matrix, accommodating volume changes and maintaining the electronic conductivity of PbO. The composite exhibits superior electrochemical performance, with a capacity of 420 mAh g-1 at a current density of 165 mA g−1, compared with previously reported Pb and PbO composite anodes. The developed anode exhibits >90% capacity retention after 9500 cycles, beginning from the second cycle, at a current density of 5.5 A g−1. Key word: gold nanoparticle、lithium ion battery、CT contrast agent、sodium ion battery II.

(4) 近年來螢光導引手術使用分子影像探針的技術已經大幅的進步,可以精準地來切除腫的位置.此篇我們合成出螢光奈米粒子(金和參雜銪的釓氧化物)結合標靶適體 AS-1411.此種奈米粒子可以有好的水溶性、生物相容性、可見螢光、和應在斷層掃瞄時有不錯的 X 光吸收能力(金)並有好的磁化率可應用在核磁共振裡 (參雜銪的釓氧化物).螢光奈米粒子可以拿來應用在斷層掃描中以老鼠進行實驗當作顯影試劑,進一步得知腫瘤的位置.更進一步的是我們可以利用奈米粒發出的螢光讓肉眼輕易地直接看到來進行切除.經由切除下來的腫瘤,我們進行 IVIS 的測試發現,其螢光強度遠大於對照組.所以可以清楚的說明從螢光圖上螢光奈米粒子可以被用來當作標靶癌症腫瘤的螢光標籤.我們的目標就是讓功能化的奈米粒子成功地成為有潛力應用在臨床上螢光導引的手術中.而結果顯示,螢光奈米粒子不僅可以當作醫藥的顯影試劑,未來也可以當作自體螢光探針來應用在手術中. 奈米粒子不僅可以應用在生醫應用中,也可以拿來用在能量儲存,像是锂電磁. 近年來科學家試著用矽來取代電極中的碳材來改善電池效能.最好的情形下可以改善將近 10 倍儲存電容量.但矽在幾次充放電過後會產生結構上的崩壞,使電池死亡.而新型以矽為電極材料的锂離子電池就是克服在充放電後所導致的電容量損失所發展出來的.此篇研究裡,我們拿矽與參雜 SPA(5-sulfoisophthalic acid)的聚苯胺做結合來當锂離子電池的電極.此一複合材料在經過一千圈的充放電後還有 99.6%的庫倫效率並高達 925 mAh g-1.代表經由參雜 SPA 可以有效改善電極強度.這個發現開啟了一個讓矽成為電極的可能.除了矽我們還使用了以碳為基材的氧化鉛複合物來嘗試作為電池,首先 PVP 經由水熱法碳化成含碳基材,再來氧化鉛參雜銅元素來增加導電度.此一材料和其他報導過含鉛材料相比有不錯的電容量(420 mAh g-1),在 5.5 A g−1 電流下可以充達 9500 圈還有大於 90%的電容量. 關鍵字: 金奈米粒子、鋰離子電池、斷層掃描顯影劑、鈉離子電池.

(5) 總目錄 Abstract……………………………………………………………………………..I 總目錄………………………………………………………………………………III Part I. Fluorescence-Guided Probes of Aptamer-Targeted Gold Nanoparticles with Computed Tomography Imaging Accesses for in Vivo Tumor Resection 1.1 Abstract…………………………………………………………………………1 1.2 Introduction…………………………………………………………………….1 1.3 Experiment Section…………………………………………………………….4 1.3.1 Preparation of Fluorescent Gold Nanoparticles……………………...4 1.3.2 Conjugation of Fluorescent Gold Nanoparticles with Meglumine Diatrizoate……………………………………………………………….5 1.3.3 Synthesis of DA-AuNPs Conjugated with Aptamer………………….6 1.3.4 Characterization Techniques…………………………………………..6 1.3.5 Cell Viability Assays of AS1411-DA-AuNPs by MCF-7 Cells……….7 1.3.6 Confocal Imaging of AS1411-DA-AuNPs Targeted to CL1-5 Cells …………………………………………………………………………………7 1.3.7 CT Imaging of AS1411-DA-AuNPs in Mice…………………………..8. 1.4 Result and Discussion………………………………………………………….8 1.4.1 Characterization of AS1411-DA-AuNPs……………………………...8 1.4.2 Optical Properties of AS1411-DA-AuNPs…………………………….10 1.4.3 Cell Viability Assays of AS1411-DA-AuNPs…………………………..11 1.4.4 Cell Targeting and Imaging……………………………………………12. III.

(6) 1.4.5 In Vitro CT Imaging of AS1411-DA-AuNPs…………………………13 1.4.6 In Vivo CT Imaging to Detect CL1-5 Tumor Location……………..14 1.4.7 AS1411-DA-AuNPs for Intraoperative Fluorescence-Guided Resection in Mouse Model………………………………………………………...15 1.4.8 Targeting Enhancement Analysis of AS1411-DA-AuNPs…………..16. 1.5 Conclusion……………………………………………………………………..18 1.6 References……………………………………………………………………...19. Part II AS1411 Aptamer-Conjugated Gd2O3:Eu Nanoparticles for Target-Specific CT/MR/Fluorescence Molecular Imaging 2.1 Abstract………………………………………………………………………..25 2.2 Introduction…………………………………………………………………...26 2.3 Experiment Section…………………………………………………………...29 2.3.1 Synthesis of A-GdO:Eu Nanoparticles……………………………….29 2.3.2 Characterization of A-GdO:Eu Nanoparticles……………………....31 2.3.3 Quantitative Polymerase Chain Reaction Protocol for A-GdO:Eu Nanoparticles……………………………………………………………31 2.3.4 Cell Viability Assays of MCF-7 Cells after Incubation with A-GdO:Eu Nanoparticles……………………………………………………………32 2.3.5 Confocal Imaging of A-GdO:Eu Nanoparticles Targeted to CL1-5 Cells ……………………………………………………………………………32. IV.

(7) 2.4 Result and Discussion………………………………………………………….33 2.4.1 Structural Characterization of A-GdO:Eu Nanoparticles…………..33 2.4.2 Optical Properties of A-GdO:Eu Nanoparticles…………………...35 2.4.3 Magnetic Properties of A-GdO:Eu Nanoparticles………………...36 2.4.4 CT Imaging and HU Measurements………………………………..37 2.4.5 Cell Viability Assays of A-GdO:Eu Nanoparticles…………………38 2.4.6 Cell Targeting and Imaging…………………………………………39 2.4.7 CT Imaging of A-GdO:Eu Nanoparticles in Mice…………………40 2.4.8 Fluorescence Imaging of A-GdO:Eu Nanoparticles into Tumor -Bearing Mice…………………………………………………………41 2.5 Conclusion……………………………………………………………………...42 2.6 References………………………………………………………………………43. Part III Highly stable cycling of lead oxide/copper nanocomposite as an anode material in lithium ion battery 3.1 Abstract…………………………………………………………………………52 3.2 Introduction…………………………………………………………………….52 3.3 Experiment Section…………………………………………………………….56 3.4 Result and Discussion………………………………………………………….58 3.5 Conclusion……………………………………………………………………...68 3.6 References………………………………………………………………………70. V.

(8) Part IV Chemical Doped Polyaniline Coating on Silicon Nanoparticles as High performance Lithium Ion Battery Anode 4.1 Abstract…………………………………………………………………………75 4.2 Introduction…………………………………………………………………….75 4.3 Experiment Section…………………………………………………………….78 4.3.1 Synthesis of SiNPs@PANi/SPA composite........................................78 4.3.2 Synthesis of SiNPs@PANi/Cl composite...........................................78 4.3.3 Electrode fabrication…………………………………………………79 4.3.4 Cell packing and electrochemical test………………………………79 4.3.5 Characterizations…………………………………………………….80 4.4 Result and Discussion…………………………………………………………..80 4.5 Conclusion………………………………………………………………………89 4.6 References……………………………………………………………………….90. VI.

(9) Fluorescence-Guided Probes of Aptamer-Targeted Gold Nanoparticles with Computed Tomography Imaging Accesses for in Vivo Tumor Resection. Abstract Recent development of molecular imaging probes for fluorescence-guided surgery has shown great progresses for precisely determining tumor margin to execute the tissue resection. Here we synthesize the fluorescent gold nanoparticles conjugated with diatrizoic acid and nucleolin-targeted AS1411 aptamer. The nanoparticle conjugates exhibit high water-solubility, good biocompatibility, visible fluorescence and strong X-ray attenuation for computed tomography (CT) contrast enhancement. The fluorescent nanoparticle conjugates are applied as a molecular contrast agent to reveal the tumor location in CL1-5 tumor-bearing mice by CT imaging. Furthermore, the orange-red fluorescence emitting from the conjugates in the CL1-5 tumor can be easily visualized by the naked eyes. After the resection, the IVIS measurements show that the fluorescence signal of the nanoparticle conjugates in the tumor is greatly enhanced in comparison to that in the controlled experiment. Our work has shown potential application of functionalized nanoparticles as a dual-function imaging agent in clinical fluorescence-guided surgery.. Introduction The development of fluorescent probes for the precise cellular targeting and imaging is of importance for cancer diagnosis and surgery in current clinical practices1-9. The functionalized fluorescent probe could be applied as a contrast agent for real-time visualization of the molecular edge between cancer and adjacent normal. 1.

(10) tissue, and consequent identification of the adequate tumor margin before surgery10-12. Great efforts have been made to develop new functionalized fluorescent probes using various types of inorganic nanoparticles such as quantum dots, and fluorescent silica spheres during the last decade13-17. The biomedical applications of these fluorescent nanoparticles have been mostly focused on long-term cellular imaging and tracking, but very few on cancers targeting at the whole organism level18-20. Of recent, gold nanoparticles with stable fluorescent emission have been successfully synthesized by the careful control of their size and surface modification21-23. In comparison to traditional organic fluorophores and fluorescent inorganic nanoparticles such as quantum dots for imaging applications, fluorescent gold nanoparticles could provide a high degree of flexibility in terms of functional groups for coating and targeting24, 25. By the attachments on the nanoparticle surface with antibody, peptide or aptamer, the resulting fluorescent gold nanoparticle conjugates can be used as molecular imaging agents for specific cell targeting26-28. In addition, the high photostablility, nontoxicity and biocompatibility made fluorescent gold nanoparticles for long-term imaging and tracking in live cells and animals29, 30. Gold atoms can induce a strong X-ray attenuation because of the high electron density (19.32 g/cm3), that makes gold nanoparticles an ideal contrast agent in computed tomography (CT) in lieu of the conventional iodine-based contrast agents31, 32. . In the medical practices, the conventional iodine-based contrast agents usually. have several drawbacks such as short imaging time, and low specificity33, 34. Thus, the design of multi-functionalized gold nanoparticles as contrast agents for precise CT molecular imaging could be of significance for cancer theranostics. Gold nanoparticles attached with antibody, peptide or aptamer on their surface have been used as contrast agents in CT imaging for the identification of cancer cells35-37. For examples, the attenuation coefficient enhancement of the molecularly targeted by gold 2.

(11) nanoparticles conjugated with UM-A9 antibodies was over 5 times than that of the identical but untargeted head and neck cancer cells or for normal cells38. Bombesin peptide functionalized gold nanoparticles were selectively targeted to the cancer receptors overexpressed in prostate, breast and small-cell lung carcinoma resulted in the enhancement of CT attenuation39. By functionalizing the surface of gold nanoparticles with the aptamer that recognizes the prostate-specific membrane antigen (PSMA), the aptamer-conjugated gold nanoparticles showed over 4-fold greater CT intensity for a targeted LNCaP cell than that of a non-targeted PC3 cell40. Those previous developments of gold nanoparticles as CT molecular contrast agents have brought meaningful new impacts on the cancer diagnosis by facilitating early detection and improving diagnostic accuracy. Recently, the technique of fluorescence-guided surgery has entered the surgical theater to help operators decide which tissues need to be resected and which tissues need to be preserved during surgery41, 42. These achievements could result in a great improvement on patient outcome and reduction of entire healthcare costs. Some progresses have been made on the uses of green fluorescent protein (GFP) to selectively and accurately label tumors and then to perform the resection under fluorescence guidance43. To the best of our knowledge, none of example has demonstrated the studies of metal or inorganic nanoparticles in in vivo fluorescent-guided resection. In addition to the uses in CT imaging as mentioned above, fluorescent gold nanoparticles could be further developed as optical imaging contrast agents to realize fluorescence-guided surgery for tumor treatments in medical surgery practices. However, one of the major challenges was the precise delivery of the fluorescent probes onto targeted cancer cells in vivo in order to clearly identify the location of tumor by CT imaging and also to provide the real time fluorescent visualization during the resection practices. 3.

(12) In this work, AS1411 aptamer with the specific targeting function to nucleolin was chosen. Fluorescent gold nanoparticles conjugated with diatrizoic acid and AS1411 aptamer (AS1411-DA-AuNPs) were synthesized and well characterized. The biocompatible AS1411-DA-AuNPs were evaluated for cancer-targeting molecular fluorescence imaging in a MCF-7 cell line by confocal microscopy. Moreover, their potential applications in medical imaging and fluorescence-guided surgery were investigated in mice. The AS1411-DA-AuNPs were injected into CL1-5 tumor-bearing mice via tail vein to detect the CL1-5 tumor location using animal micro-CT modality and then the fluorescence enhancement of the nanoparticles was further examined on resected CL1-5 tumor with in vivo imaging system (IVIS). To evaluate the targeting efficiency, the orange-red fluorescence signals coming from the AS1411-DA-AuNPs (with aptamer) in CL1-5 tumors were compared with the control experiment, which the fluorescent nanoparticles without the aptamer conjugation was applied. Our work has demonstrated a new concept that fluorescent gold nanoparticles could. be. applied. as. contrast. agents. for. imaging. and. target-specific. fluorescence-guided surgery in the future clinical practices.. Experiment Section Preparation of Fluorescent Gold Nanoparticles The fluorescent gold nanoparticles were prepared by a one-pot green method with some modifications49. Briefly, 15 mL of HAuCl4 aqueous solution (1 % w/w) was added to 15 mL of 25 mM L-glutathione aqueous solution under vigorous stirring. The color of HAuCl4 and L-glutathione solution changed from transparent to dark brown and finally became transparent. The HAuCl4 and L-glutathione solution was then heated to 40 °C with vigorous stirring in the dark environment for 3~5 days.. 4.

(13) After 3~5 days, the fluorescent gold nanoparticle solution was obtained. The solution was subsequently purified by centrifuging at 15000 rpm for 5 min. The supernatant solution was kept after removal of the particles in the bottom. The supernatant was further precipitated by adding ethanol (the volume ratio between supernatant and ethanol was 1:2) and the yellow cloudy mixture was formed. Afterward, the mixture was centrifuged at 18000 rpm for 10 min. A precipitate of fluorescent gold nanoparticles was formed at the bottom of the centrifuge tube. After the removal of supernatant solution in the centrifuge tube, the precipitate of fluorescent gold nanoparticles was redispersed in deionized water with 80 mg N-methyl-D-glucamine by sonication. The purified fluorescent gold nanoparticles were stored at 4 °C in the dark environment for the following experiments. The fluorescent gold nanoparticle solution was stable more than 6 months. Conjugation of Fluorescent Gold Nanoparticles with Meglumine Diatrizoate 0.5 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (1.151 g) and 0.5 M N-hydroxysuccinimide (NHS) (1.553 g) were prepared in 20 mL 2-(N-morpholino) ethanesulfonic acid (MES) (0.1M), respectively. First, 302.4 mg meglumine diatrizoate was dissolved in 10 mL deionized water. Second, 750μL of 0.5 M EDC and NHS were sequentially added in meglumine diatrizoate solution. The mixture was stirred at room temperature for 1 h. Third, fluorescent gold nanoparticle solution was added in the mixture and then was stirred at room temperature for 12 h. The solution of fluorescent gold nanoparticles conjugated with diatrizoic acid (DA-AuNPs) was washed with 20 mL ethanol and then centrifuged at 18000 rpm for 10 min. The pellet of the DA-AuNPs was redispersed in deionized water. Finally, the DA-AuNPs were collected and filtrated by 0.22 μm syringe filter (Millipore MILLEX® MCE membrane) to remove larger nanoparticles. 5.

(14) Synthesis of DA-AuNPs Conjugated with Aptamer The aqueous solution of DA-AuNPs was dried under vacuum. The dried DA-AuNPs were dispersed in PBS buffer solution (87.5 mg/mL) for the following experiments. 300 μL of 100 μM AS1411 aptamer was added in a 20 mL flask50. And then 30 μL of 5 mM EDC and NHS were sequentially added in the flask. 200 μL of 87.5 mg/mL DA-AuNPs was also added in the flask. At this time, the flask was added with 10 mL deionized water and the reactants were stirred at 4 °C for 2 h. After 2 h reaction, the AS1411-DA-AuNPs solution was added with the same volume ethanol and then centrifuged at 18000 rpm for 5 min. The supernatant solution was slowly removed without disturbing the precipitate. The precipitate was dissolved in deionized water. Afterward, the AS1411-DA-AuNPs aqueous solution was dried under vacuum. The dried AS1411-DA-AuNPs were redispersed in PBS buffer solution and finally stored at 4°C for the following experiments. Characterization Techniques Instruments. of. transmission. electron. microscope. (TEM). and. X-ray. energy-dispersive spectroscopy (EDX) were performed on a Philips/FEI Tecnai 20 G2 S-Twin transmission electron microscope. A small amount of AS1411-DA-AuNPs was dispersed in deionized water by sonicator. A drop of AS1411-DA-AuNPs solution was placed on an amorphous carbon membrane supported by a copper grid. The copper grid was characterized by TEM and EDX. The ligand attachment of fluorescent gold nanoparticles was characterized by FTIR spectrophotometer (JASCO 200E FT-IR) with the resolution of 2 cm-1. UV-vis absorption and fluorescence spectroscopy was measured by using PerkinElmer HP-8453 and AMINCO Bowman Series 2 spectrometers, respectively, with the resolution of 1 nm.. 6.

(15) Cell Viability Assays of AS1411-DA-AuNPs by MCF-7 Cells Dulbecco’s Modified Eagle Medium (DMEM) containing 3.7 g/L sodium bicarbonate, 1 % penicillin, and 10 % fetal bovine serum was used to maintain human breast cancer cell line MCF-7. The MCF-7 cell culture was placed in a 37 °C humidified atmosphere with 5 % CO2, and DMEM was refreshed every 3 days. For cell viability assays of AS1411-DA-AuNPs, MCF-7 cells were first seeded onto 96-well plates at a density of 6000 cells per well. After 100 μL of DMEM was added to each well, the 96-well plates were returned to incubators for cell attachment about 12 h. Following cell attachment, each well was added with another 100 μL of AS1411-DA-AuNPs solution for 24 h. After the removal of supernatant, the MCF-7 cells were then treated with 20 μL of MTT (5 mg/mL in PBS) and then incubated for 4 h. Finally, the medium was eliminated, and the cells were lysed with DMSO (100 μL). The absorbance of the purple formazan was recorded at 570 nm by Nanodrop spectrophotometer. Confocal Imaging of AS1411-DA-AuNPs Targeted to CL1-5 Cells The human lung adenocarcinoma cell line CL1-5 was cultured in 25 cm2 flask with RPMI-1640 medium containing 10 % (v/v) fetal bovine serum at 37 °C under 5 % CO2 environment. After several days, the CL1-5 cells were put on quartz bottom dishes and incubated for 24 h. Before the incubation with the fluorescent nanoparticle conjugates, the CL1-5 cells were washed with PBS solution (pH =7.4). For the comparison of the specific targeting efficiency of the fluorescent nanoparticle conjugates with and without AS1411 aptamer on their surface, AS1411-DA-AuNPs and DA-AuNPS (100 ng/mL) were respectively incubated with CL1-5 cells at 37 °C for 30 min. Afterward, the CL1-5 cells were washed with PBS buffer solution for three times and then the CL1-5 cells were further fixed with 4 % paraformaldehyde for 10 min at the room temperature. Hoechst 33258 (Invitrogen, 0.5 μg/mL) was. 7.

(16) treated for 5 min to stain the CL1-5 cell nucleus. Confocal fluorescence microscopy was carried out with an Ultra-View RS confocal system (Perkin Elmer, Wellesley, MA). CT Imaging of AS1411-DA-AuNPs in Mice All the animal studies were performed according to the protocols approved by the Laboratory Animal Center, Academia Sinica. 8-week old NOD-SCID mice (BioLasco, Taiwan) were subcutaneously inoculated with 2 X 106 CL1-5 cells by using a 23-gauge needle. The length (L) and width (W) of tumors were measured with calipers and the tumor volumes were calculated as LW2/2. After 20 days, tumor volumes were about 400 mm3. Mice were randomly divided into two groups: mouse A (n = 3) and mouse B (n = 3). AS1411-DA-AuNPs and DA-AuNPs (100 μL, 370 mg/mL) were intravenously injected into mouse A and mouse B, respectively. The CT signal intensity was measured and calculated by micro-CT (SkyScan1076, Bruker) at different time intervals (0, 0.5, 2, 4 and 24 h) after the injection. The measurement of CT signal before injection was used as baseline for HU value calculation.. Results and Discussion Characterization of AS1411-DA-AuNPs The sequential synthetic steps of AS1411-DA-AuNPs were described in Figure 1a. In brief, the synthesis of fluorescent gold nanoparticles conjugated with diatrizoic acid and AS1411 aptamer consists of three steps. First, the fluorescent gold nanoparticles were prepared via one-pot green method. Second, the fluorescent gold nanoparticles were conjugated with commercial iodine-based contrast agent of diatrizoic acid (DA-AuNPs) to increase CT sensitivity and water solubility. Finally, DA-AuNPs were modified with AS1411 aptamer (AS1411-DA-AuNPs) using EDC activation reaction. The conjugation of fluorescent gold nanoparticles with diatrizoic acid (DA-AuNPs) 8.

(17) was first studied by Fourier transform infrared spectroscopy before they were conjugated with AS1411 aptamer. They exhibited the characteristic IR bands of primary amine salt (N-H stretch; 2929.88 and 3047.97 cm-1) and amide (N-H stretch; 3291.51 cm-1) as shown in Figure 1b. Also, the characteristic N-H stretch band of amide shifted from 3291.51 to 3409.59 cm-1. The IR data confirmed that, after the EDC/NHS coupling reaction, the carboxyl group of diatrizoic acid was bound with primary amine of glutathione on fluorescent gold nanoparticles to form amide bond and the IR absorption of primary amine salt was disappeared. To further verify the successful conjugation of diatrizoic acid onto the surface of gold nanoparticle, the EDX analysis of DA-AuNPs were applied, and the results showed that the atomic percentages of the gold, iodine and sulfur were 14.32, 5.41, and 13.79 %, respectively (Figure 1c). The detection of iodine also implied that diatrizoic acid was successfully attached on gold nanoparticle surface. The resulting DA-AuNPs exhibited high water solubility. Afterward, DA-AuNPs were then conjugated with nucleolin-targeted AS1411 aptamer using EDC/NHS activation reaction in aqueous solution. After the conjugation of AS1411 and DA-AuNPs, the extra AS1411 aptamer was removed thoroughly from the surface of AS1411-DA-AuNPs. The quantitative polymerase chain reaction (qPCR) of AS1411-DA-AuNPs was performed to calculate the quantity of AS1411 aptamer conjugated onto DA-AuNPs. The concentration of the aptamer of AS1411-DA-AuNPs solution was found to be 99.7 μM. The efficiency of DA-AuNPs conjugated with AS1411 aptamer was calculated to be ~66.46 % via EDC/NHS activation reaction. Overall, the qPCR result suggested that the DA-AuNPs were efficiently conjugated with AS1411 aptamer. After all, the water-soluble AS1411-DA-AuNPs were examined by TEM to determine their structural properties (Figure 1d). 9.

(18) Figure 1 (a) The sequential synthetic steps of AS1411-DA-AuNPs. (b) The FTIR spectra of fluorescent gold nanoparticles (red) and DA-AuNPs (blue). (c) EDX analysis of AS1411-DA-AuNPs. (d) TEM image of AS1411-DA-AuNPs. The nanoparticles exhibited nearly spherical shape with the average size of 2.4±0.4 nm based on the averaged sizes of 200 nanoparticles in TEM images.. Optical Properties of AS1411-DA-AuNPs Figure 2 shows the UV-vis absorption and fluorescence emission spectra of AS1411-DA-AuNPs. The disappearance of the surface plasmon absorption of fluorescent AS1411-DA-AuNPs at ~520 nm could be due to the high oxidation states of extra small gold nanoparticles (~2.4 nm in diameter) and the lack of free electrons to provide free electrons to generate the coherent oscillations44, 45. The fluorescence spectrum of AS1411-DA-AuNPs in Figure 2 showed clear orange-red emission with the maximum at 620 nm. Several studies suggested that the emission of AS1411-DA-AuNPs could be induced from ligand-metal charge transfer on thiolate capped gold nanoparticles (~2 nm), which the electrons were transferred from the sulfur atom of surface glutathione to the Au core46-48. Notably, the distinguishable 10.

(19) observable orange-red color of high water-soluble AS1411-DA-AuNPs was readily seen by naked eyes under irradiation of a hand-held long-wave UV lamp. This characteristic makes AS1411-DA-AuNPs a candidate for fluorescent molecular imaging.. Figure 2 UV-vis absorption spectrum (black) and emission spectrum (red) of AS1411-DA-AuNPs. The emission spectrum of AS1411-DA-AuNPs was measured with the excitation of 400 nm wavelength. The quantum yield of orange-red emission from AS1411-DA-AuNPs was measured to be 1.1 % using the fluorescence of R6G as the standard. Cell Viability Assays of AS1411-DA-AuNPs The cytotoxicity measurements were evaluated in a MCF-7 cell line by MTT assay to reveal the biocompability of AS1411-DA-AuNPs. The assay was performed over a dosage range of 10-3~1 mg/mL of AS1411-DA-AuNPs solutions. In Figure 3, the MTT assay showed no significant cytotoxic response (the cell viability >80 %) detected even at the concentration of 1 mg/mL, which was much higher than that usually used for live-cell imaging studies by confocal microscopy. This study indicated that the AS1411-DA-AuNPs have shown very low cytotoxicity. We therefore explore their applications as a molecular contrast agent.. 11.

(20) Figure 3 Cytotoxicity evaluated by MTT assay in the range of 1-1000 μg/mL of AS1411-DA-AuNPs. The MCF-7 cells were incubated with AS1411-DA-AuNPs solution for 24 h.. Cell Targeting and Imaging To study the potential of the nanoparticles as fluorescent probes for biomedical imaging, the CL1-5 cells were separately incubated with DA-AuNPs (without aptamer attached) and AS1411-DA-AuNPs (with aptamer attached) for the comparison. After 30 min, the cells of both samples were compared under a confocal microscope. As shown in Figure 4a, the fluorescence of AS1411-DA-AuNPs (red pseudocolor) was clearly observed with high signal-to-background ratio in the CL1-5 cells incubated with AS1411-DA-AuNPs. In contrast, under the same imaging condition, no detectable fluorescence of DA-AuNPs was observed in the CL1-5 cells incubated with DA-AuNPs (Figure 4b). The result showed that the fluorescent AS1411-DA-AuNPs, but not DA-AuNPs, entered efficiently into the CL1-5 cells. Thus, the AS1411 aptamer of AS1411-DA-AuNPs leaded to the specific binding to nucleolin highly expressed CL1-5 cells in the plasma membrane. Overall, our experiments clearly demonstrated that AS1411-DA-AuNPs exhibited the. 12.

(21) capability as fluorescent probes for cancer targeting.. Figure 4 Two-dimensional confocal microscopic images of CL1-5 cells (a) incubated with AS1411-DA-AuNPs and (b) incubated with DA-AuNPs. The red and blue pseudocolors represent the fluorescent signal of AS1411-DA-AuNPs and the nucleus (stained with Hoechst 33258), respectively.. In Vitro CT Imaging of AS1411-DA-AuNPs Next, we explored the possibility of using AS1411-DA-AuNPs as a contrast agent in CT imaging. The CT signal intensity of AS1411-DA-AuNPs was measured at the weight concentrations in range of 10 to 40 mg/mL. The CT signal intensity in Figure 5 revealed positive contrast enhancement in a dose-dependent manner. Also, when the weight concentration changed from 10 to 40 mg/mL, the signal intensity of AS1411-DA-AuNPs exhibited much higher CT signal intensity than that of the commercially available iodine-containing agents (Omnipaque, Ge Healthcare) at the same concentration. The strong CT signal intensity of AS1411-DA-AuNPs was mainly generated from the high X-ray absorption coefficient of gold and iodine (X-ray absorption coefficient at 100 keV, Au: 5.16 cm2/g; I: 1.94 cm2/g).. 13.

(22) Figure 5 The CT values of AS1411-DA-AuNPs and Iohexol (Omnipaque). The insets showed the CT images, the respective weight concentrations of AS1411-DA-AuNPs (above the image), and the respective CT values (below the image).. In Vivo CT Imaging to Detect CL1-5 Tumor Location The in vivo molecular imaging was studied on the CL1-5 tumor-bearing mice. The target-specific AS1411-DA-AuNPs (370 mg/mL, 100 μL) were injected through tail vein into the CL1-5 tumor-bearing mice. The CT imaging was performed in the mice using animal micro-CT modality (SkyScan1076, Bruker) to identify the location of tumor. The CT images were acquired before and after injection of the AS1411-DA-AuNPs. At 30 min post injection, a distinct 106 % more contrast enhancement of the CL1-5 tumor targeted with AS1411-DA-AuNPs was measured in CL1-5 tumor-bearing mouse (marked by yellow circle in Figure 6). The CT image showed clearly that AS1411-DA-AuNPs were predominantly accumulated in the bladder, which later excreted out of the mice rapidly. The animal experiments showed that the AS1411-DA-AuNPs were able to be the molecular contrast agent to detect the tumor location in mice using CT modality. After the detection of CL1-5 tumor location by CT imaging, the tumor-bearing mice were sacrificed. The CL1-5 tumor 14.

(23) targeted with AS1411-DA-AuNPs in the tumor-bearing mice was further examined under ultraviolet light irradiation to demonstrate the applications as intraoperative fluorescent imaging probes in guided surgery.. Figure 6 The CT image of the CL1-5 tumor-bearing mouse was taken at 30 min post injection of AS1411-DA-AuNPs. The location of CL1-5 tumor was marked by yellow circle.. AS1411-DA-AuNPs for Intraoperative Fluorescence-Guided Resection in Mouse Model The fluorescence imaging was performed under a handy ultraviolet light (4W) in Figure 7. The orange-red fluorescence coming from AS1411-DA-AuNPs was readily observed in the CL1-5 tumor (marked by yellow circle) and can be seen by naked eyes. The fluorescence of AS1411-DA-AuNPs was further used for the identification of the CL1-5 tumor margin. The tissue with orange-red fluorescence was resected and the normal tissue without orange-red fluorescence was preserved in the tumor-bearing mouse. Thus, the CL1-5 tumor targeted with AS1411-DA-AuNPs was successfully taken out by intraoperative fluorescence-guided resection. This result has demonstrated the great potentials of using AS1411-DA-AuNPs as a molecular imaging probe in fluorescent-guided surgery. In comparison to most currently used. 15.

(24) organic molecules, AS1411-DA-AuNPs are able to provide long-term imaging times, high photostability, multiple imaging functions and feasible surface modifications with specific-targeted molecules. However, in order to quantitatively evaluate the specific targeting efficiency of AS1411-DA-AuNPs, the further experiments on fluorescent signal measurements by IVIS were performed next.. Figure 7 The fluorescence image of CL1-5 tumor-bearing mouse at 30 min post injection of AS1411-DA-AuNPs. The yellow circle points out the location of the CL1-5 tumor.. Targeting Enhancement Analysis of AS1411-DA-AuNPs Two groups of CL1-5 tumor-bearing mice, A and B, were separately injected via tail vein with AS1411-DA-AuNPs (with AS1411 aptamer) and DA-AuNPs (without aptamer). At 30 min post-injection, the mouse A and B were sacrificed and then the CL1-5 tumors in the mouse A and B were taken out. Figure 8a (top panel) showed that the CL1-5 tumors were observed under white light. The color and size of both CL1-5 tumors were similar. However, under excitation by ultraviolet light, the CL1-5 tumor of mouse A injected with AS1411-DA-AuNPs revealed orange-red fluorescence as shown in Figure 8a (bottom panel), whereas no red fluorescence was observed in. 16.

(25) the CL1-5tumor of mouse B injected with DA-AuNPs. Those images in Figure 8a suggested that the AS1411-DA-AuNPs have been specifically targeted into the nucleolin highly expressed CL1-5 tumor, and the fluorescent nanoparticles could be potentially used as intraoperative fluorescence imaging probes during guided resection. To further quantitatively analyze the targeting efficiency, IVIS imaging was utilized to compare the fluorescent intensity of the CL1-5 tumors of mouse A and mouse B. The IVIS image in Figure 8b (right) demonstrated that the strong fluorescence was observed in the CL1-5 tumor targeted with AS1411-DA-AuNPs of mouse A. The fluorescent intensities, which were calculated with the IVIS image, ranged from 1.88x106~2.89x107 p/sec/cm2/sr. However, with the control experiment of DA-AuNPs injection, the fluorescence in the CL1-5 tumor of mouse B was not detected at the background level. Furthermore, the total photo flux in CL1-5 tumors was measured to evaluate the enhancements of fluorescence with mouse A and mouse B (Figure 8c). The total photon fluxes in the CL1-5 tumors are 1.98x108 and 1.15x108 p/sec/cm2/sr with mouse A and mouse B, respectively. The fluorescent enhancement of CL1-5 tumor with targeting AS1411-DA-AuNPs injection of mouse A was 172 % in comparison to non-targeting DA-AuNPs injection of mouse B. Overall, the in vivo experiments in Figure 8 have showed that the AS1411 aptamer conjugated with fluorescent gold nanoparticles can be used for selectively targeting nucleolin highly expressed CL1-5 tumor, and the strong fluorescence of AS1411-DA-AuNPs was sufficient to contrast the tumor lesions.. 17.

(26) Figure 8 (a) CL1-5 tumors under white light (top panel) and ultraviolet light (bottom panel).. CL1-5. tumors. incubated. with. DA-AuNPs. (left. column). and. AS1411-DA-AuNPs (right column). (b) The IVIS images of CL1-5 tumors incubated with DA-AuNPs (left) and AS1411-DA-AuNPs (right). The color scale is an indication of the fluorescent intensity from red (low) to blue (high). (c) The total photon fluxes calculated from CL1-5 tumors shown in Figure 8b.. Our in. vivo studies. on. CL1-5. tumor-bearing. mice. showed. that. the. AS1411-DA-AuNPs have applied as a molecular contrast agent to detect the CL1-5 tumor. location. by. CT. imaging.. The. orange-red. fluorescence. emitting. from AS1411-DA-AuNPs in CL1-5 tumor has been easily seen by the naked eyes in the tumor-bearing mice. And, the CL1-5 tumor was successfully resected from the mouse under visible fluorescence guiding. With the target-specific guiding of florescent gold nanoparticle (AS1411-DA-AuNPs), the fluorescence signal of resected CL1-5 tumor was enhanced by 172 % compared to that of DA-AuNPs by IVIS measurements.. Conclusion Overall, our experiments have established a simple CT/fluorescent imaging platform using gold nanoparticle conjugates for tumor imaging and resecting in a mice model. The platform has demonstrated several advantages in comparison to other molecular imaging systems. First, besides the lung cancer in a mouse model. 18.

(27) studied in this work, our platform could be also applicable in other tumor systems. Since the synthetic procedures of this platform were relatively simple, a fluorescent molecular probe could be easily prepared by the conjugation of DA-AuNPs with specific targeted aptamers (antibodies or peptides) to perform molecular imaging. Secondly, AS1411-DA-AuNPs exhibit safe and simple chemical properties to avoid the complexities of tumor-selective gene delivery in comparison to the green fluorescent protein that was recently developed as the fluorescent imaging agent in the field of fluorescence-guided surgery. Third, in our platform, the CT and fluorescence images were simultaneously obtained, allowing us to double check the targeted tumor information such as the location, shape and size. Therefore, much comprehensive diagnostic information could be provided using AS1411-DA-AuNPs as a contrast agent, and the precise resection of the tumor could become feasible under fluorescence-guided surgery. Finally, the platform has offered a simple method to determine tumor margin based on the images from IVIS measurements after the tumor resection. However, we understand that, in our experiment, the correlation between fluorescence intensity and the amount of tumor cells should be further analyzed quantitatively. In the future, other analytical methods for in vivo tumor cell determination should be applied to make a comparison with the results obtained from IVIS measurements. Thus, the florescent intensity from IVIS measurements could be transformed to be much quantitative information in order to create a clear guideline on the determination of tumor margins. Eventually, we expect that our platform based on fluorescent gold nanoparticle conjugates will become a powerful tool in the field of fluorescence-guided surgery. References 1.. Yang, H. et al. Protein conformational dynamics probed by single-molecule electron transfer. Science 302, 262-266 (2003).. 19.

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(33) AS1411 Aptamer-Conjugated Gd2O3:Eu Nanoparticles for Target-Specific CT/MR/Fluorescence Molecular Imaging. Abstract The europium-doped gadolinium oxide (Gd2O3:Eu) nanoparticles have been synthesized, and then their surface have been conjugated with nucleolin-targeted AS1411 aptamer to form functionalized target-specific Gd2O3:Eu nanoparticles (A-GdO:Eu nanoparticles). The A-GdO:Eu nanoparticles present strong fluorescence in the visible range, high magnetic susceptibility, X-ray attenuation and good biocompatibility. The A-GdO:Eu nanoparticles have been applied to test molecular expression of nucleolin highly expressed CL1-5 lung cancer cells under a confocal microscope. The fluorescent imaging clearly reveals that the nanoparticles can be applied as fluorescent tags for cancer-targeting molecular imaging. Furthermore, taking together the results of their excellent T1 contrast and strong CT signal, the A-GdO:Eu nanoparticles have been demonstrated a great capability to use as the dual modality contrast agent for CT and MR molecular imaging. The animal experiments also show that the A-GdO:Eu nanoparticles are sufficient to contrast the tissues of BALB/c mice using CT modality. Moreover, the obvious red fluorescence of A-GdO:Eu nanoparticles can be visualized in the tumor by naked eyes. Overall, our results demonstrate that the A-GdO:Eu nanoparticles could not only be served as new medical contrast agents but also as intraoperative fluorescent imaging probes for guided surgery in the near future.. 25.

(34) Introduction Recent developments for the synthesis of functionalized nanoparticles have made great progresses for medical imaging applications1-5. Different types of metal and metal oxide nanoparticle conjugates have been designed as a contrast agent for various medical imaging systems such as computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET). 6-13. . The. advantage of using nanoparticles as an imaging agent in comparison to most current organic molecules is that they are able to provide sharp images, long-term imaging time, high photostability and multiple imaging functions. 14,15. . In particular, the. functionalized nanoparticles with multiple physical and chemical properties such as superparamagetism, strong X-ray sensitivity and radioactivity have provided great advantages to develop the new contrast agents for simultaneous uses in two different imaging systems. 16-20. . Those agents are expected to offer more comprehensive. diagnostic information during the medical practice. For examples, we have recently synthesized anti-Her2 antibody conjugated FePt nanoparticles used as CT/MRI dual molecular imaging contrast agents. 21. . The location of the mice MBT2 tumors was. preciously revealed by both imaging techniques. Lee et al. also reported that Fe3O4/TaOx core/shell nanoparticles were observed using CT and MRI revealed the high and low vascular regions of the tumor 22. Beside the metal alloyed nanoparticles, dextran-superparamagnetic iron oxide nanoparticles coated with. 64. CuII-complexes. were detected in draining lymph nodes by PET and MR dual imaging. 23. . Also,. 18. F. labeled cross-linked dextran iron oxide nanoparticles were prepared for the dynamic PET/CT dual imaging24. Those examples have demonstrated that with dual imaging techniques, the resulting image has the benefits to reduce the screening time and to enhance the imaging interpretation by overcoming the limitations of single imaging modality.. 26.

(35) Fluorescent tags have gradually become important biological imaging agents in live cells and animal models for revealing the specific biological activity even to assist medical surgery. 25-28. . In medical applications, the fluorescence also provides great. advantages to discriminate between tumor and normal tissue and consequentially to determine an adequate tumor-free margin. Recently, the fluorescent tags of the nanoparticles such as quantum dots, gold nanoparticles, nanodiamond and yttrium oxides have made significant developments to provide the real time visualization of the tumor in the intraoperative image guided surgery. 29-36. . Several examples have. demonstrated that the fluorescent nanoparticles were used for fluorescent image guided in the animal model especially for imaging of tumor tissues, organs and sentinel lymph nodes 37-40. They have successfully shown that the fluorescence of the nanoparticles. was. clearly. observed. in. the. animal. model. with. high. signal-to-background ratio. Also, the extensive studies on in vivo toxicity revealed that the fluorescence nanoparticles did not affect normal functioning of organs. The fluorescent nanoparticle-based tags are very promising probes for the applications spanning from primary tumor detection to further tumor staging in research and clinic. Thus, it will be quite important that the nanoparticles could offer the possibility to put the images right under the hands of the surgeon, if possible, also to give the high resolution 3D tomography information of the tissues and the lesion supplied by CT/MRI. One of the great advantages of using nanoparticles as imaging agents is that it is chemically feasible to attach specific molecules such as antibodies, aptamers, peptides and small molecules onto the nanoparticle surface. 41,42. . Therefore, the new. biomolecule conjugated nanoparticles could also bring a chance to develop specific-targeted molecular imaging agents 43. Recently, many new synthetic aptamers have been discovered with high affinity and specificity onto various types of cancer 27.

(36) cells. The aptamers exhibited low molecular weights, lack of immunogenicity, and ready availability provide significant advantages for targeted molecular imaging of cancer cells. Concurrently, the aptamer-functionalized nanoparticles have been applied as a molecular imaging agent for the cell recognization using current imaging techniques. Gold nanoparticles functionalized with a prostate-specific membrane antigen RNA aptamer were allowed to detect the targeted binding of prostate cancer cells by clinical CT instrumentation. 44. . In fluorescence imaging, aptamer AS1411. modified polymeric nanoparticles were used as a molecular contrast agent to observe the brain glioma. 45. . Cobalt-ferrite conjugated with aptamer AS1411 nanoparticles. were observed in the tumor-bearing mice at C6 tumor sites by MRI modality 46. With advances in surface modification of nanoparticles and aptamer selection technologies, aptamer-functionalized nanoparticles were being explored as promising molecular contrast agents for diagnostic applications. To further combine the current imaging techniques, i.e. CT and MRI, the aptamer-functionalized nanoparticles could be developed as a dual-modal and even tri-modal molecular imaging contrast agent. One of potential candidates will be gadolinium oxide nanoparticles that have been reported as a possible multimodal imaging contrast agent previously. 47-49. . However,. to the best of our knowledge, the developments of Gd2O3 nanoparticles as the trimodal imaging agents for CT, MR, and also fluorescence imaging are still few. More importantly, the design and synthesis of Gd2O3 nanoparticles with specific cancer targeting function are still lacking. In this work, the aptamer AS1411 conjugated europium-doped gadolinium oxide (A-GdO:Eu) nanoparticles were synthesized and explored for their potential as a trimodal molecular contrast agent for CT, MR and fluorescence imaging(Figure).. 28.

(37) A series of comprehensive evaluations of transmission electron microscope (TEM),. X-ray diffractometer,. UV-vis. spectrophotometer. and. fluorescence. spectrophotometer were performed to characterize their morphology, structure, optical property. Magnetic susceptibility, T1 weighted imaging and X-ray attenuation of A-GdO:Eu nanoparticles were investigated by superconducting quantum interference device (SQUID), MRI and CT, respectively. The biocompatibility of A-GdO:Eu nanoparticles was evaluated in a MCF-7 cell line by MTT assay. Furthermore, the fluorescent A-GdO:Eu nanoparticles were then incubated with CL1-5 cells to demonstrate the cancer-targeting molecular fluorescence imaging by confocal microscopy. Also, the nanoparticles were injected into mice to demonstrate the CT contrast enhancement using animal micro-CT modality. The fluorescence of A-GdO:Eu nanoparticles was further examined in the tumor-bearing mice. These results provided an insight for further developments of multimodal molecular contrast agent in the applications of medical imaging and surgery in the near future.. Experiment Section Synthesis of A-GdO:Eu Nanoparticles Europium-doped gadolinium oxide (Gd2O3:Eu) nanoparticles were synthesized via the polyol method according to the literatures. First, 5.7 mmol GdCl3 ·6H2O and 1.1 mmol EuCl3 ·6H2O were dissolved in 30 mL of diethylene glycol. After strong. 29.

(38) stirring for 30 min, 1 mL of 3 M aqueous NaOH solution was added and then the mixture was heated at 140 ℃ for 1 h in order to complete dissolution of the compounds. Second, the mixture was heated at 180 ℃ for 4 h under vigorous stirring in refluxing diethylene glycol. Afterward, the mixture changed to transparent suspension. The transparent suspension was filtered with a 0.22 μm membrane (poly-ethersulfone) to remove any large sized agglomeration. Finally, the Gd2O3:Eu nanoparticles capped with diethylene glycol were obtained. The capping layer on Gd2O3:Eu nanoparticles were further exchanged from the diethylene glycol to citric acid. The filtered suspension was heated to 150 ℃ under stirring and then 0.5 mmol NaOH with 0.5 mmol citric acid dissolved in 2 mL diethylene glycol were added. The resulting solution was then refluxed at 180 ℃ for 30 min under vigorous stirring. The changes of the solution were observed from transparent to white. The white solution was washed by addition methanol followed by centrifugation for several times to get white precipitation. After drying the white precipitation under vacuum, a powder form of the citric acid-capped Gd2O3:Eu nanoparticles were obtained. The citric acid-capped Gd2O3:Eu was further functionalized with aptamer following the procedures as described previously.[46] The aptamer AS1411 (5’-TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’) was prepared, and then the citric acid-capped Gd2O3:Eu nanoparticles were linked to a 5’-NH2-modified AS1411 aptamer using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC). In this work, 10 mg of the powder form citric acid-capped Gd2O3:Eu nanoparticles were redispersed in 1 mL phosphate-buffered saline (PBS) solution. The solution of citric acid-capped Gd2O3:Eu nanoparticles was added to equivalent of EDC and AS1411 aptamer (1, 2, 5, 6, 7, 8 and 10 μmol) under stirring at 4 ℃. Within 1 h, the solution containing the AS1411 aptamer conjugated with the citric acid-capped Gd2O3:Eu nanoparticles 30.

(39) (abbreviated as A-GdO:Eu nanoparticles) was prepared. The solution containing A-GdO:Eu nanoparticles was centrifuged at 13000 rpm for 15 min under 4 ℃ and washed in PBS solution twice. Finally, the A-GdO:Eu nanoparticles were redispersed in the PBS solution before the next steps. Characterization of A-GdO:Eu Nanoparticles Transmission electron microscope (TEM) and X-ray energy-dispersive spectroscopy (EDX) was carried out on a Philips/FEI Tecnai 20 G2 S-Twin transmission electron microscope. A small amount of A-GdO:Eu nanoparticles was redispersed in deionized water by sonicator. A drop of A-GdO:Eu nanoparticles solution was placed on an amorphous carbon membrane supported by a copper grid. The copper grid was characterized by TEM and EDX. Powder X-ray diffraction data was collected on a Bruker D8 Advance diffractometer. The powder of A-GdO:Eu nanoparticles was placed on amorphous Si wafer and the workup procedure was carried out with Cu Kα radiation. (λ. =1.54178. Å ).. Magnetic. measurements. were. performed. by. superconducting quantum interference device (SQUID) magnetometer (MPMS, Quantum Design). The measurements were recorded between -50000 and 50000 Oe at 5 K. Quantitative Polymerase Chain Reaction Protocol for A-GdO:Eu Nanoparticles The method of quantitative polymerase chain reaction (qPCR) was performed to calculate the quantity of aptamer of A-GdO:Eu nanoparticles. To obtain the standard curve, the aptamer solution of 100 μM was diluted in 10, 1, 0.1, and 0.01 μM, respectively. Deionized water was used as negative control. Primer sequences of forward primer (AAG GAG GGG CAA GCA AC) and reverse primer (GGT TTT TTT TTT TTT TTT TTT T) were designed in this work. By following the introduction of commercial qPCR reagent, 1 μL of A-GdO:Eu nanoparticles was added to SYBR Green 5 μL, forword primer 0.25 μL (10 μM), reverse primer 0.25 μL 31.

(40) (10 μM), template 1 μL and deionized water 3.5 μL. The qPCR conditions were 35 cycles by the sequential steps of 95 ℃ (10min), 95 ℃ (30 s), 48 ℃ (30 s), 72 ℃ (20 s). Dissociation stage conditions were designed by the sequential steps of 95 ℃ (15 s), 60 ℃ (15 s) and 95 ℃ (15 s). The values of Ct were used to calculate the number of aptamer conjugated with A-GdO:Eu nanoparticles by the method of linear interpolation with standard curve. Cell Viability Assays of MCF-7 Cells after Incubation with A-GdO:Eu Nanoparticles Human breast cancer cell line MCF-7 was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 3.7 g/L sodium bicarbonate, 1 % penicillin, and 10 % fetal bovine serum. The culture was placed in a 37 °C humidified atmosphere with 5% CO2, and the medium was changed every 3 days. After A-GdO:Eu nanoparticles were added into the MCF cells, the cell viability was tested by MTT assay. MCF-7 cells were seeded onto 96-well plates at a density of 5000 cells per well. Each well was added to 100 μL of DMEM. The plates were then returned to incubators for 12 h for cell attachment. Afterward, the powder of A-GdO:Eu nanoparticles were redispersed in deionized water. For cell viability assay, each well was added with another 100 μL of A-GdO:Eu nanoparticles solution for 24 h. After the removal of supernatant, the cells were then treated with 20 μL of MTT (5 mg/mL in PBS) and then incubated for 4 h. Afterward the medium was removed, and the cells were lysed with 100 μL of DMSO. Finally, the absorbance of the purple formazan was recorded at 570 nm. Confocal Imaging of A-GdO:Eu Nanoparticles Targeted to CL1-5 Cells The human lung adenocarcinoma cell line CL1-5 was cultured in 25 cm2 flask in RPMI-1640 medium supplemented with 10 % (v/v) fetal bovine serum at 37 ℃ 32.

(41) under 5 % CO2. After several days, the cells were put on quartz bottom dishes and incubated for 24 h. Before the incubation with the Gd2O3:Eu nanoparticles, cells were washed with PBS (pH =7.4). To test for the specific targeting of nucleolin, two samples of 100 ng/mL Gd2O3:Eu nanoparticles with and without AS1411 nucleolin aptamer conjugation were separately incubated with CL1-5 cells at 37 °C for 30 min. After washing with PBS buffer, the CL1-5 cells were fixed with 4 % paraformaldehyde for 10 min at the room temperature. Hoechst 33258 (Invitrogen) of 0.5 μg/mL was treated for 5 min to stain the CL1-5 cell nucleus. Confocal fluorescence imaging was recorded by an Ultra-View RS confocal system (Perkin Elmer, Wellesley, MA).. Results and Discussion Structural Characterization of A-GdO:Eu Nanoparticles The syntheses of the aptamer conjugated europium-doped gadolinium oxide (A-GdO:Eu) nanoparticles involved three major steps. First, Gd2O3:Eu nanoparticles were prepared via the polyol method. Second, the capping layer of Gd2O3:Eu nanoparticles was replaced from diethylene glycol to citric acid for further surface modification with aptamer. Finally, the A-GdO:Eu nanoparticles were prepared by the conjugation of the citric acid-capped Gd2O3:Eu nanoparticles with aptamer using EDC activation. The average size, shape, and crystallinity of A-GdO:Eu nanoparticles were examined by TEM. The A-GdO:Eu nanoparticles are approximately of a spherical shape as shown in the image of Figure 1a. The average size of A-GdO:Eu nanoparticles was estimated to be ~10 nm from the averaged sizes of 100 nanoparticles in TEM images. The chemical compositions of A-GdO:Eu nanoparticles were analyzed with EDX and ICP-MS. The atomic ratio of Eu/Gd of the nanoparticles was found to be ~0.19 based on both the EDX and ICP-MS analyses. The value 33.

(42) agreed well with the ratio of Eu/Gd in the initial reactants. The crystal structure and purity of A-GdO:Eu nanoparticles were examined by XRD. As shown in Figure 1b, all the strong diffraction peaks of A-GdO:Eu nanoparticles were indexed to the cubic phase of Gd2O3 (JCPDS card No. 11-604). The main peaks of A-GdO:Eu nanoparticles at 2θ = 28.15°, 32.37°, 46.09° and 57.04° are indexed to (222), (400), (440) and (622), respectively. The nanoparticles exhibited well crystallinity and no characteristic peak of Eu oxides was detected. The diffraction pattern of A-GdO:Eu nanoparticles was similar to that of Gd2O3 nanoparticles. The slightly differences of the peak position and intensity of A-GdO:Eu nanoparticles were observed because of Eu doping. In addition, The average size (~10.3 nm in diameter) of A-GdO:Eu nanoparticles was calculated from Debye-Scherrer equation based on the full width at half-maximum of the (400) reflection peak. The results agreed with the observation in TEM image of Figure 1a.. Figure 1. (a) TEM image of A-GdO:Eu nanoparticles. (b) The XRD scans of Gd2O3 and A-GdO:Eu nanoparticles.. The quantitative polymerase chain reaction (qPCR) was performed to calculate the quantity of aptamer conjugated with A-GdO: Eu nanoparticles. The concentration of. 34.

(43) aptamer was calculated to be 7.475 μM of A-GdO:Eu nanoparticles solution. The result indicated that the A-GdO:Eu nanoparticles were successfully obtained by the conjugation of the citric acid-capped Gd2O3:Eu nanoparticles with aptamer using EDC activation. The A-GdO:Eu nanoparticles were then designed for the applications in the cancer-targeting molecular imaging. Optical Properties of A-GdO:Eu Nanoparticles The optical properties were studied by UV-vis absorption and photoluminescence spectroscopy. Figure 2a shows the UV-vis spectra of the Gd2O3 and A-GdO:Eu nanoparticles. The absorption band at ~278 nm was the characteristic peak of A-GdO:Eu nanoparticles after Eu atoms were doped into Gd2O3 nanoparticles. The band was attributed to the charge-transfer absorption between the O2- and Eu3+ [51, 52]. Upon excitation at 278 nm, the emission spectrum exhibited five groups of emission lines at about 580, 593, 616, 654, and 698 nm (Figure 2b), which were induced from the 5D0-7FJ (J = 0, 1, 2, 3, 4) transitions of Eu3+ [53]. Because the strong red fluorescence of A-GdO:Eu nanoparticles under the excitation were distinguishable from green autofluorescence of tissues, the nanoparticles could be potentially used as the fluorescent imaging agents.. Figure 2. (a) UV-vis absorption spectra of Gd2O3 and A-GdO:Eu nanoparticles. (b) Emission spectra of A-GdO:Eu nanoparticles excited at 278 nm. 35.