金纳米红外光敏与稀土上转换荧光材料光学性质与生物应用
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摘要
贵金属，稀土掺杂荧光上转换等红外光功能纳米材料在肿瘤靶向治疗，活体荧光成像和传感等生物医学应用领域展示了诱人的前景，但在实际应用中还存在一定的障碍和局限。金纳米棒在近红外区具有强表面等离子共振特性，可有效将吸收的光转化成热，是红外肿瘤热疗的理想光敏材料；然而，金纳米棒表面性质影响纳米粒子在溶液环境中的存在状态尤其是肿瘤治疗中靶向功能的实现。稀土红外-可见上转换纳米材料在活体成像中具有穿透深度深，可避免生物体干扰信号等优点，但其荧光上转换效率较低，且可见光发射在活体成像中难于在活体中直接探测。为解决以上问题，本论文以金纳米棒与稀土掺杂上转化纳米粒子为研究对象，一方面，合成了新的乳腺癌细胞SK-BR-3的靶向肽，并将其与聚丙烯酸(PAA)修饰的金纳米棒共价连接，应用于乳腺癌细胞（SK-BR-3）选择性光热治疗研究；采用新的表面层谷胱甘肽修饰金纳米棒，探讨了谷胱甘肽修饰金纳米棒与蛋白质的相互作用，并研究叶酸、罗丹明功能化金纳米棒的靶向成像及光热治疗作用；另一方面，比较了NaYF_4:Yb~(3+),Er~(3+)与NaLuF_4:Yb~(3+),Er~(3+)的结构与发光性质，确定了上转换发光与基质构成、晶体结构和尺寸的依赖关系；在此基础上选择结构易于调控的NaYF_4:Yb~(3+),Tm~(3+)作为纳米荧光体，利用其近红外上转换发射与金纳米棒间的荧光共振能量传递诱导的荧光猝灭检测亲和素蛋白。取得的主要成果有：
[1]利用层层包覆法（Layer-by-Layer）制备了PAA修饰的金纳米棒，高分子量PAA修饰的金纳米棒在复杂溶液中具有更好的稳定性且无细胞毒性；通过噬菌体展示肽库技术成功获得与HER2阳性细胞特异性结合的12肽模拟物，模拟肽功能化的金纳米棒能够选择性靶向乳腺癌细胞（SK-BR-3）且具有优良的光热治疗效果。
[2]通过配体交换作用即强健的Au-S键形成制备谷胱甘肽修饰的金纳米棒，修饰的金纳米棒不仅在酸、碱性缓冲液且在高浓度盐离子缓冲液中均具有非常好的稳定性；最重要的是，与蛋白质的非特异性吸附减弱；经过叶酸与罗丹明功能化的金纳米棒具有更好的靶向成像及光热治疗效果。
[3]利用高温溶剂热法在不同反应温度合成NaREF_4（RE=Lu/Y）；在NaREF_4合成过程中，用Lu3+取代Y3+后，NaLuF_4纳米晶生长更快，且更易形成立方相结构；NaLuF_4与NaYF_4荧光强度变化主要与纳米晶的尺寸、结构密切相关，与Lu/Y本身基质关系不大；提出一种新的物理模型解释了NaREF_4纳米晶尺寸、结构及浓度依赖的颜色变化性质。
[4]利用水热法合成水溶性、近红外响应的PEI-NaYF_4:Yb~(3+),Tm~(3+)；通过共价键将生物素分子连接到荧光纳米粒子表面，且通过静电吸附将亲和素连接到聚丙烯胺（PAH）修饰的金纳米棒表面；根据纳米粒子之间荧光共振能量转移产生的荧光猝灭检测亲和素浓度，提供了一种新型的近红外响应的荧光传感系统。
The infared functional nanomaterials such as noble metal and rare earth dopedupconversion luminescent nanoparticles have shown attractive prospects in tumortarget therapy, living fluorescence imaging, sensing and other biomedical applications.However, there are still existing some obstacles and limitations in practice. Strongsurface plasmon resonance properties of gold nanorods in near infrared region makeeffective light-heat conversion, which make gold nanorods being ideal photosensitivematerials in tumor hyperthermia. However, surface properties of gold nanorodsinfluence their stability, especially, the targeting function in tumor therapy in complexenvironment. With the advantages of deep penetration and low backgroundfluorescence, Rare-earth infared-visible upconversion luminescent nanoparticles haveapplied in living imaging, but the low fluorescence conversion efficiency makesvisible light emission be difficult to be detected in vivo imaging. To solve the aboveproblems, based on gold nanorods and rare earth doped upconversion luminescentnanoparticles in this paper, one hand, we modified gold nanorods with PAA andconjugated targeting peptide on the rods surface, and the prepared targetingconjugates were applied in selective photothermal thrapy; then, we discussed theinteraction between glutathione modified gold nanorods and protein, and studied thetargeting imaging and photothermal therapy of folic acid and rhodamine B conjugatedgold nanorods; the other hand, we compared the synthesis and optical properties ofNaLuF_4:Yb~(3+), Er~(3+)and NaYF_4:Yb~(3+), Er~(3+)and confirmed the dependencies ofupconversion luminescence with substrate composition, crystal structure and size;based on above, we chose the easily controlled NaYF_4:Yb, Tm as upconversionluminescence nanocrystalline and detected adivin concentration through fluorescentquenching between gold nanorods and NaYF_4:Yb, Tm. The results we obtained were shown as follows:
[1] We prepared PAA modified gold nanorods (GNRs) through Layer-by-Layermethod and observed the higher weight of PAA modified GNRs with betterbiocompatibility and non-cytotoxity. And then we obtained targeting peptide throughphase display peptide library technology, which could specific interact with HER2positive cells. Finally, we obtained selective photothermal therapy with targetingpeptide functionalized GNRs.
[2] We prepared glutathione modified gold nanorods (GSSG-GNRs) throughrobust Au-S bond information and demonstrated the GSSG-GNRs were stable inaqueous solution over a broad range of pH and high ionic strength values.Importantly, the glutathione coating greatly reduces non-specific protein adsorptionon GNRs. Then we obtained selective targeting imaging and photothermal therapywith folic acid and rhodamine B functionalized GNRs.
[3] We synthesized NaREF_4:Yb~(3+), Er~(3+)(RE=Lu, Y) at different reactiontemperatures by the solvothermal method. In the NaREF_4preparation process, whenthe Y3+was replaced by Lu3+, NaLuF_4nanocrystals grew faster and were easier toform a cubic phase structure. The fluorescence intensity change of NaLuF_4andNaYF_4was closely related with the size and structure of the nanocrystalline and hadlittle to do with Lu/Y-matrix. On emphasis, we gave a universal explanation on thephase, and size dependent color-tuning behaviors of NaREF_4:Yb~(3+), Er~(3+)(RE=Y, Lu).
[4] We prepared hydrophilic PEI coating NaYF_4: Yb, Tm nanoparticles throughhydrothermal method. The biotion and adivin molecules were conjugated to PEIcoating NaYF_4and PAH modified gold nanorods through covalent bonds,respectively. Adivin concentration was detected based on fluorescence resonanceenergy transfer (FRET) between upconversion nanopaticles and gold nanorods. Andwe provided a novel near-infared responsive sensor system.
[1]利用层层包覆法（Layer-by-Layer）制备了PAA修饰的金纳米棒，高分子量PAA修饰的金纳米棒在复杂溶液中具有更好的稳定性且无细胞毒性；通过噬菌体展示肽库技术成功获得与HER2阳性细胞特异性结合的12肽模拟物，模拟肽功能化的金纳米棒能够选择性靶向乳腺癌细胞（SK-BR-3）且具有优良的光热治疗效果。
[2]通过配体交换作用即强健的Au-S键形成制备谷胱甘肽修饰的金纳米棒，修饰的金纳米棒不仅在酸、碱性缓冲液且在高浓度盐离子缓冲液中均具有非常好的稳定性；最重要的是，与蛋白质的非特异性吸附减弱；经过叶酸与罗丹明功能化的金纳米棒具有更好的靶向成像及光热治疗效果。
[3]利用高温溶剂热法在不同反应温度合成NaREF_4（RE=Lu/Y）；在NaREF_4合成过程中，用Lu3+取代Y3+后，NaLuF_4纳米晶生长更快，且更易形成立方相结构；NaLuF_4与NaYF_4荧光强度变化主要与纳米晶的尺寸、结构密切相关，与Lu/Y本身基质关系不大；提出一种新的物理模型解释了NaREF_4纳米晶尺寸、结构及浓度依赖的颜色变化性质。
[4]利用水热法合成水溶性、近红外响应的PEI-NaYF_4:Yb~(3+),Tm~(3+)；通过共价键将生物素分子连接到荧光纳米粒子表面，且通过静电吸附将亲和素连接到聚丙烯胺（PAH）修饰的金纳米棒表面；根据纳米粒子之间荧光共振能量转移产生的荧光猝灭检测亲和素浓度，提供了一种新型的近红外响应的荧光传感系统。
The infared functional nanomaterials such as noble metal and rare earth dopedupconversion luminescent nanoparticles have shown attractive prospects in tumortarget therapy, living fluorescence imaging, sensing and other biomedical applications.However, there are still existing some obstacles and limitations in practice. Strongsurface plasmon resonance properties of gold nanorods in near infrared region makeeffective light-heat conversion, which make gold nanorods being ideal photosensitivematerials in tumor hyperthermia. However, surface properties of gold nanorodsinfluence their stability, especially, the targeting function in tumor therapy in complexenvironment. With the advantages of deep penetration and low backgroundfluorescence, Rare-earth infared-visible upconversion luminescent nanoparticles haveapplied in living imaging, but the low fluorescence conversion efficiency makesvisible light emission be difficult to be detected in vivo imaging. To solve the aboveproblems, based on gold nanorods and rare earth doped upconversion luminescentnanoparticles in this paper, one hand, we modified gold nanorods with PAA andconjugated targeting peptide on the rods surface, and the prepared targetingconjugates were applied in selective photothermal thrapy; then, we discussed theinteraction between glutathione modified gold nanorods and protein, and studied thetargeting imaging and photothermal therapy of folic acid and rhodamine B conjugatedgold nanorods; the other hand, we compared the synthesis and optical properties ofNaLuF_4:Yb~(3+), Er~(3+)and NaYF_4:Yb~(3+), Er~(3+)and confirmed the dependencies ofupconversion luminescence with substrate composition, crystal structure and size;based on above, we chose the easily controlled NaYF_4:Yb, Tm as upconversionluminescence nanocrystalline and detected adivin concentration through fluorescentquenching between gold nanorods and NaYF_4:Yb, Tm. The results we obtained were shown as follows:
[1] We prepared PAA modified gold nanorods (GNRs) through Layer-by-Layermethod and observed the higher weight of PAA modified GNRs with betterbiocompatibility and non-cytotoxity. And then we obtained targeting peptide throughphase display peptide library technology, which could specific interact with HER2positive cells. Finally, we obtained selective photothermal therapy with targetingpeptide functionalized GNRs.
[2] We prepared glutathione modified gold nanorods (GSSG-GNRs) throughrobust Au-S bond information and demonstrated the GSSG-GNRs were stable inaqueous solution over a broad range of pH and high ionic strength values.Importantly, the glutathione coating greatly reduces non-specific protein adsorptionon GNRs. Then we obtained selective targeting imaging and photothermal therapywith folic acid and rhodamine B functionalized GNRs.
[3] We synthesized NaREF_4:Yb~(3+), Er~(3+)(RE=Lu, Y) at different reactiontemperatures by the solvothermal method. In the NaREF_4preparation process, whenthe Y3+was replaced by Lu3+, NaLuF_4nanocrystals grew faster and were easier toform a cubic phase structure. The fluorescence intensity change of NaLuF_4andNaYF_4was closely related with the size and structure of the nanocrystalline and hadlittle to do with Lu/Y-matrix. On emphasis, we gave a universal explanation on thephase, and size dependent color-tuning behaviors of NaREF_4:Yb~(3+), Er~(3+)(RE=Y, Lu).
[4] We prepared hydrophilic PEI coating NaYF_4: Yb, Tm nanoparticles throughhydrothermal method. The biotion and adivin molecules were conjugated to PEIcoating NaYF_4and PAH modified gold nanorods through covalent bonds,respectively. Adivin concentration was detected based on fluorescence resonanceenergy transfer (FRET) between upconversion nanopaticles and gold nanorods. Andwe provided a novel near-infared responsive sensor system.
引文
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