摘要
背景早期诊断和治疗是肿瘤研究中的关键科学问题。纳米科技的快速发展为解决这一关键科学问题带来了新机遇。具有独特等离子共振效应的金纳米棒粒子有潜力应用于肿瘤的早期诊断和治疗。
目的本研究将利用树形分子取代金纳米棒表面的毒性分子十六烷基三乙基溴化铵(CTAB),然后与精氨酸-甘氨酸-天冬氨酸多肽(RGD)偶联构建生物相容性好的靶向肿瘤细胞和肿瘤血管内皮细胞的生物探针,并解决在病变局部选择性治疗的理论问题,旨在为黑素瘤的早期诊断和治疗提供新的策略。
方法首先应用“晶种生长”法合成均一的金纳米棒粒子(GNRs),然后利用巯基改性的G4.0聚酰胺胺(PAMAM)树形分子替代金纳米棒表面的CTAB分子。PAMAM树形分子修饰的纳米粒子与生物配体RGD多肽通过共价键偶联得到具有靶向功能的生物探针(RGD-dGNRs)。利用高分辨率透射电镜、原子力显微镜、核磁共振波谱和紫外-可见吸收光谱等仪器对纳米探针进行表征。
选择人脐静脉血管内皮细胞(HUVEC)、黑素瘤A375和乳腺癌MCF-7等细胞系来研究纳米探针的靶向性。应用CCK-8方法评价RGD-dGNRs纳米探针对细胞的毒性作用。利用暗视野显微镜来观察RGD-dGNRs纳米探针与肿瘤细胞结合的敏感性和特异性。同时设立严格的竞争抑制结合实验来推测细胞的结合位点。当肿瘤细胞与RGD-dGNRs纳米探针结合后,应用与金纳米棒纵向等离子共振吸收峰相近的近红外(NIR)808nm激光照射,能量分别采用30 mW,70 mW,110 mW,150 mW四个等级,光斑直径为1.0 mm,照射时间为4分钟。应用Trypan blue(台盼蓝)染色和Calcein-AM染色评价纳米探针吸收激光转化为热能后杀伤肿瘤细胞的效能。
将5×106黑素瘤细胞注射到裸鼠右侧大腿皮下,当肿瘤直径达到5mm左右,200μg的RGD-dGNRs纳米探针从尾静脉注入小鼠体内,然后分别在3h, 6h, 9h和12h四个时间点处死3只小鼠,收集血液、肿瘤组织以及心、肝、肾等重要脏器,用电感偶合质谱仪(ICP-Mass)测定各个脏器和肿瘤组织的金纳米棒含量,评价RGD-dGNRs纳米探针在动物体内的分布状况。为推测RGD-dGNRs纳米探针与细胞结合的位点,同时进行竞争抑制结合实验,在注射RGD-dGNRs纳米探针前,给予小鼠过量的RGD多肽(0.5mg)。
30只荷瘤小鼠被随机分为三组,分别为实验组(200μg RGD-dGNRs +NIR激光照射);PBS对照组(PBS+NIR激光照射);空白对照组(未予以任何处理)。纳米探针从小鼠静脉注射6小时后,肿瘤部位予以NIR的808nm激光照射,功率密度24 W/cm2,光斑直径5mm,照射时间5分钟。每周治疗1次,共治疗4次。应用皮肤反射式共聚焦显微镜(RCM)观察肿瘤的组织学和血流变化。通过观察肿瘤体积变化和Kaplan-Meier生存曲线来观察RGD-dGNRs光热治疗对小鼠生存期的影响。结果通过“晶种生长法”制备的金纳米棒(GNRs-CTAB)大小均一,面径比为4.2左右,横向等离子共振吸收峰在520 nm,纵向等离子共振吸收峰在821nm左右。树形分子修饰后的金纳米棒(dGNRs)显示了很好的分散性。RGD-dGNRs纳米探针的纵向等离子共振吸收峰发生了轻度红移(3nm)。RGD-dGNRs纳米探针在生理环境下显示了很好的分散性和稳定性。金纳米棒探针在200ug/ml浓度范围内未见明显的细胞毒性作用,而GNRs-CTAB在浓度在50μg/ml左右即可显示明显的细胞毒性作用。
在暗视野显微镜观察下,A375黑素瘤细胞和HUVEC细胞与RGD-dGNRs纳米探针结合后显示明亮的金黄色,不能被水冲洗掉。过量的RGD多肽明显抑制了RGD-dGNRs纳米探针与黑素瘤细胞的结合。另外RGD-dGNRs纳米探针并不与αvβ3整合素阴性乳腺癌细MCF-7胞结合。
A375黑素瘤细胞与RGD-dGNR纳米探针结合30分钟后,在70mW的NIR激光照射下,可见部分细胞被杀死,当激光能量达到110mW时,细胞死亡明显增加。dGNRs纳米粒子孵育或单纯的NIR激光照射并未导致细胞明显死亡。RGD-dGNRs纳米探针与αVβ3整合素阴性的MCF-7乳腺癌细胞孵育,然后在NIR激光照射下,未见明显的细胞死亡。细胞的杀伤情况与RGD-dGNR纳米探针的浓度也密切相关。在110m的激光能量照射下,当RGD-dGNR纳米探针的浓度增加到100ug/ml时,几乎所有的黑素瘤细胞被杀死。因此RGD-dGNRs体外光热治疗的最佳浓度可能为100μg/ml。
药代动力学实验显示,随着时间的延长,RGD-dGNRs纳米探针在肿瘤部位的蓄积逐渐增加。6小时后,RGD-dGNRs纳米探针在肿瘤部位可见明显的蓄积,约占金纳米棒总量的17%。过量的RGD多肽几乎完全抑制了RGD-dGNRs纳米探针在肿瘤部位的蓄积。与空白对照组和PBS治疗组相比,实验组小鼠的肿瘤体积在整个观察期间明显变小(P﹤0.01),其中四只小鼠的肿瘤组织几乎完全消失。空白对照组小鼠的平均生存期大约为3周,PBS治疗组的小鼠平均生存期大约为4周,而实验组小鼠的平均生存期大于7周,组间有显著性差异(P =0.006)。Kaplan-Meier曲线也显示RGD-dGNRs纳米探针的光热治疗能明显延长小鼠的生存时间。
RCM实时观察到RGD-dGNRs光热治疗荷瘤小鼠3小时后,肿瘤部位血流明显减少,而对照组小鼠的肿瘤组织血流无明显改变。治疗7周后,RCM观察到RGD-dGNRs纳米探针光热治疗小鼠的肿瘤组织内出现大量的网状或树枝状胶原纤维束,组织学HE染色显示为大量胶原纤维增生,类似疤痕结构。而RCM在对照组小鼠的肿瘤组织内可见大量折射增强的细胞,部分细胞聚集成簇,其对应的HE染色可见大量的黑素瘤细胞。
结论本研究成功合成了生物相容好的RGD-dGNRs纳米探针,这些新颖的纳米探针能准确识别活体肿瘤细胞与肿瘤中的新生血管内皮细胞。在NIR激光照射下,纳米探针吸收光能转化为热能将肿瘤血管破坏和肿瘤细胞杀伤。这种纳米复合物探针也适用于其他αvβ3整合素阳性的肿瘤细胞。这些智能化的生物探针在未来的肿瘤分子成像与光热治疗方面具有潜在的应用价值。
Backgrounds There is currently an increasing need for early detection and treatment of cancer before anatomic anomalies are visible. Nanotechnology has advanced greatly in recent years is becoming a promising approach for cancer diagnosis and treatment. One of the current challenges in biomedicine is to develop safe and effective nanoprobes for tumor targeting and selective therapy. Gold nanorods (GNRs) are strongly absorbing in at near-infrared wacelength and have excellent potential for photothermal therapy.
Objective Here, we prepared unique tumor targeting nanoprobes by conjugating dendrimer-modified GNRs with RGD peptides, with the aim of developing a novel method for early detection, diagnosis and imaging of melanoma.
Methods First, gold nanorods were prepared by seed-mediated surfactant directed approach and controlling the different reaction synthetic conditions.Then, we used the partially thiolated polyamidoamine (PAMAM) dendrimer to replace CTAB molecules on the surface of GNRs, with the aim of improving the biocompatibility of prepared GNRs. Finally, we prepared unique tumor targeting nanoprobes by conjugating dendrimer-modified GNRs with RGD peptides. Prepared RGD-dGNR nanoprobes were characterized by high-resolution-transmission-electron microscopy (HR-TEM), atomic force microscopy (AFM), 1H Nuclear magnetic resonance (1H-NMR) and UV-vis absorbance spectroscopy.
HUVEC cell line with overexpression ofαvβ3 integrin was used as positive control group. The MCF-7 cell line with lower expression of integrinαvβ3 was selected as the negative control group. The melanoma A375 cell line with over-expression of integrin αvβ3 was selected as the test group. The Cell Counting Kit-8 assay was used to measure cytotoxicity of synthesized nanoprobes following the instruction of the kit. Then we evaluated the specificity and sensitivity of RGD-dGNR nanoprobes for tumor cell targeting using the dark field microscope. Parallel competition inhibition control experiments were set up. Cells incubated with and without RGD-dGNR nanoprobes were exposed to NIR laser irradiation, with a wavelength of 808 nm and varying intensities, from 30 mW (4 W/cm2) to 150 mW (20 W/cm2) in increments of 40 mW. The laser spot size is 1.0 mm in diameter, and the exposure time is 4 min. The cell viability was tested by both 0.4% trypan blue and Calcein-AM staining.
Melanoma A375 cells (5×106) were injected subcutaneously into the right rear flank area of nude mice. When tumors grew to 5 mm in diameter, 200μg of RGD-dGNR nanoprobes was injected into mice via tail vein. For the blocking experiment, ten mice were injected with the mixture of 0.5 mg of RGD peptides and 200μg of RGD-dGNR nanoprobes. Mice were respectively sacrificed at 3 h, 6 h, 9 h, and 12 h. Blood and organs were collected and kept in liquid nitrogen. The amount of RGD-dGNRs was measured by inductively coupled plasma mass spectrometry.
Mice loaded with tumors were randomly divided into three groups: test group (200μg of nanoprobes plus NIR laser irradiation); sham control group (10 mice) (PBS plus NIR laser irradiation), and blank control (untreated). The nude mice were injected with 200μg of prepared nanoprobes in PBS via tail vein. At 6 h after injection, the mice were anesthetized and irradiated with a NIR laser with a wavelength of 808 nm at a power density of 24 W/cm2 and a spot size of 5 mm diameter for 5 min. Tumors were irradiated for four times per month, once every week. Real time reflectance confocal micrscopy (RCM) was used to collect images of tumors from test group and control group and to assess the histological and blood flow changes. Hematoxylin and eosin staining was used to examine tumor histological changes. Tumor size and mice survivability were monitored for 7 weeks.
Results The original gold nanorods (GNRs-CTAB) are about 42 nm in length, and 10 nm in width. Dendrimer-modified GNRs exhibit better dispersion in water solution than GNRs-CTAB. GNRs-CTAB has two absorption bands, a weak short-wavelength band around 520 nm and a strong long-wavelength band around 821 nm. RGD-dGNRs exhibit an absorption band red-shift (about 3nm). We observed that RGD-dGNRs exhibited good stability and dispersibility in water or organic solution with different pH conditions. Initial evaluation of the cytotoxicity of RGD-dGNR nanoprobes showed both the dendrimer-modified GNRs and RGD-dendrimer-modified GNRs were biologically nontoxic within the concentration of 200μg/mL. GNR-CTAB concentration of 100μg/ml exhibited marked cytotoxicity.
Under dark field microscopy, melanoma A375 cells incubated with RGD-dGNR nanoprobes exhibit a strong golden color; the melanoma A375 cells incubated with dGNRs and preincubated with free RGD peptides did not exhibit a golden color, and similar negative results also were observed for MCF-7 cells incubated with dGNRs or RGD-dGNRs, which highly suggests that free RGD peptides can block the binding of RGD-dGNR nanoprobes with integrinαvβ3 over-expressed in the tumor cells, and RGD-dGNR nanoprobes can specifically target melanoma A375 cells.
A375 cells incubated with RGD-dGNR nanoprobes exhibited destruction within the laser spots after exposure to the laser at 70 mW. When the laser energy reached 110 mW, the amount of destroyed tumor cells increased accordingly. Few damaged cells were observed for the A375 cells treated with dGNRs or NIR light alone. Similarly, no dead cells were observed for MCF-7 cells treated with RGD-dGNR nanoprobes. These results fully showed that RGD-dGNR nanoprobes were only employed to kill cancer cells with overexpression of integrinαvβ3 under NIR irradiation. Under the condition of 110 mW laser irradiation for 4 min, as the amount of RGD-dGNR nanoprobes in the medium increased, the amount of destroyed cells also increased accordingly. 100μg/ml RGD-dGNR nanoprobes was considered as the optimal photothermal therapeutic concentration for in vitro cancer cells.
The distribution of RGD-dGNR nanoprobes was examined in the whole body of mouse models, 17% of the RGD-dGNR nanoprobes accumulated in local tumor tissues at 6 h after injection. Nanoprobes in the tumor tissues increased gradually as time increased, which fully suggests that the prepared RGD-dGNR nanoprobes were targeting tumor tissues. Six hours after injection was selected as the optimal time to begin the NIR laser irradiation on the tumor locations. For the competition inhibition experiment group, few RGD-dGNR nanoprobes accumulated in the tumor location, highly indicating the free RGD peptides can first bind with integrinαvβ3 on the surface of tumor cells, and block the specific binding of RGD-dGNR nanoprobes with the tumor cells and vasculature endothelial cells in vivo.
Under the NIR laser irradiation, the average tumor size in the test group was markedly smaller than those in the control groups. Interestingly, we observed, the tumor tissues in four mice almost completely disappeared in three weeks or so. We observed that the median survivability of mice in the control group without any treatment was three weeks, in the control group treated with PBS plus NIR laser was four weeks, and in the test group was more than seven weeks. The survivability of mice in the test group was markedly stronger than the one in the control group, P= 0.006. The Kaplan-Meier curve suggests that the therapy based on nanoprobe injection plus NIR laser irradiation can markedly increase the survival time of mice with tumors.
We observed that the blood flow inside tumor tissues in the test group began to be blocked at 3 h after NIR laser irradiation using real time reflectance confocal microscopy (RCM). In the control group, no obvious dynamic blood flow alteration was observed. Additionally, after seven weeks of treatment, RCM images of tumors in the test group clearly displayed numerous gossamer-like collagen bundles and branchlike collagens. Numerous reflective melanoma cells were still distributed throughout the tumor tissues in the control group. Accordingly, histological analysis showed that the tumors in test group after seven weeks of treatment exhibited a scarlike structure containing numerous collagen bundles. A lot of tumor cells were observed in those tumors untreated and treated with the PBS plus laser irradiation.
Conclusion In conclusion, our study confirms that the RGD-conjugated dGNR nanoprobes are not cytotoxic, can specifically target tumor cells and vascular cells inside tumor tissues, and exhibit selective destructive effects on the melanoma cells under NIR laser irradiation. Compared with presently available reports, the RGD-dGNR nanoprobe-based targeting therapy possesses some major advantages, which can extend to various tumors with overexpressed integrinαvβ3. The RGD-dGNR-based therapy strategy has great potential in applications such as tumor targeting imaging and selective photothermal therapy.
引文
1 Jemal, A. et al. Cancer Statistics, 2009. CA: a cancer journal for clinicians (2009).
2孙东杰,高天文,李春英等.西安、重庆两所医院皮肤恶性黑素瘤回顾.中华皮肤科杂志37 (2004).
3 Allen, E. Malignant Melanoma. British Medical Journal 2, 1067 (1955).
4 Gray-Schopfer, V., Wellbrock, C. & Marais, R. Melanoma biology and new targeted therapy. Nature 445, 851 (2007).
5 Hood, J. & Cheresh, D. Role of integrins in cell invasion and migration. Nature reviews. Cancer 2, 91 (2002).
6 Varner, J. & Cheresh, D. Integrins and cancer. Current opinion in cell biology 8, 724 (1996).
7 Eliceiri, B. & Cheresh, D. The role of |áv integrins during angiogenesis: insights into potential mechanisms of action and clinical development. Journal of Clinical Investigation 103, 1227-1230 (1999).
8 Kumar, C. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Current drug targets 4, 123 (2003).
9 Butler, B., Williams, M. & Blystone, S. Ligand-dependent activation of integrin alpha vbeta 3. The Journal of biological chemistry 278, 5264 (2003).
10 D'Souza, S., Ginsberg, M. & Plow, E. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends in biochemical sciences 16, 246 (1991).
11 Schottelius, M., Laufer, B., Kessler, H. & Wester, H. Ligands for Mapping alpha (v) beta (3)-Integrin Expression in Vivo. Accounts of chemical research (2009).
12 Assa-Munt, N., Jia, X., Laakkonen, P. & Ruoslahti, E. Solution structures and integrin binding activities of an RGD peptide with two isomers. Biochemistry 40, 2373 (2001).
13 Maheshwari, G., Brown, G., Lauffenburger, D., Wells, A. & Griffith, L. Cell adhesion and motility depend on nanoscale RGD clustering. Journal of cell science 113, 1677 (2000).
14 Zhao, M., Wang, C., Jiang, X. & Pen, S. Synthesis of RGD containing peptides and their bioactivities. Preparative biochemistry & biotechnology 32, 363 (2002).
15 Richards, J. et al. Engineered fibronectin type III domain with a RGDWXE sequence binds with enhanced affinity and specificity to human alphavbeta3 integrin. Journal of molecular biology 326, 1475 (2003).
16 Dechantsreiter, M. et al. N-Methylated cyclic RGD peptides as highly active and selective alpha (V) beta (3) integrin antagonists. Journal of medicinal chemistry 42, 3033 (1999).
17 Liekens, S., De Clercq, E. & Neyts, J. Angiogenesis: regulators and clinical applications. Biochemical pharmacology 61, 253 (2001).
18 Tucker, G. Integrins: molecular targets in cancer therapy. Current oncology reports 8, 96 (2006).
19 Humphries, M., Akiyama, S., Komoriya, A., Olden, K. & Yamada, K. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. The Journal of cell biology 103, 2637 (1986).
20 Sivolapenko, G. et al. Imaging of metastatic melanoma utilising a technetium-99m labelled RGD-containing synthetic peptide. European journal of nuclear medicine 25, 1383 (1998).
21 von Wallbrunn, A. et al. In vivo imaging of integrin alpha v beta 3 expression using fluorescence-mediated tomography. European journal of nuclear medicine and molecular imaging 34, 745 (2007).
22 Allman, R., Cowburn, P. & Mason, M. In vitro and in vivo effects of a cyclic peptide with affinity for the alpha (nu) beta3 integrin in human melanoma cells. European journal of cancer (Oxford, England: 1990) 36, 410 (2000).
23 Ramos, O. et al. A novel alpha (v) beta (3)-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis. Clinical & experimental metastasis 25, 53 (2008).
24 Haubner, R. et al. Noninvasive Visualization of the Activated |áv|?3 Integrin in Cancer Patients by Positron Emission Tomography and [18F] Galacto-RGD. PLoS Medicine 2 (2005).
25 Haubner, R. et al. Noninvasive imaging of alpha (v) beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer research 61, 1781 (2001).
26 Chen, X. et al. 18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis. Nuclear medicine and biology 31, 179 (2004).
27 Cai, W. et al. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano letters 6, 669 (2006).
28 Smith, B. et al. Real-time intravital imaging of RGD-quantum dot binding to luminal endothelium in mouse tumor neovasculature. Nano letters 8, 2599 (2008).
29 Zavaleta, C. et al. Noninvasive Raman spectroscopy in living mice for evaluation of tumor targeting with carbon nanotubes. Nano letters 8, 2800 (2008).
30刘红洁,王.,张春丽等. 131^Ⅰ标记RGD环肽在荷瘤小鼠体内分布与显像研究.中国医学影像技术24, 3 (2008).
31李前伟,郑.,张广运等. 125I—RGD—4CY肿瘤αvβ3受体显像的实验研究.第三军医大学学报24, 1340 (2002).
32 Emerich, D. & Thanos, C. Nanotechnology and medicine. Expert opinion on biological therapy 3, 655 (2003).
33 PISSUWAN, D., VALENZUELA, S. & CORTIE, M. Prospects for gold nanorod particles in diagnostic and therapeutic applications. Biotechnol. Gen. Eng. Rev 25, 93¨C112 (2008).
34 Huang, X., Jain, P., El-Sayed, I. & El-Sayed, M. Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomedicine (London, England) 2, 681 (2007).
35 Johnson, C. J., Dujardin, E., Davis, S. A., Murphy, C. J. & Mann, S. Vol. 12 1765-1770 (Royal Society of Chemistry, 2002).
36 Jian, Z., Yong-Chang, W. & Shi-Nong, Y. Fluorescence spectrum characteristics of gold nanorods. Chinese Physics Letters 21, 559 (2004).
37 Li, C., Male, K., Hrapovic, S. & Luong, J. Fluorescence properties of gold nanorods and their application for DNA biosensing. Chemical communications (Cambridge, England), 3924 (2005).
38 Murphy, C. et al. Chemical sensing and imaging with metallic nanorods. Chemical communications (Cambridge, England), 544 (2008).
39 Johnson, C., Dujardin, E., Davis, S., Murphy, C. & Mann, S. Growth and form of gold nanorods prepared by seed-mediated, surfactant-directed synthesis. Journal of Materials Chemistry 12, 1765-1770 (2002).
40 Nikoobakht, B. & El-Sayed, M. Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Mater 15, 1957-1962 (2003).
41 Hauck, T., Ghazani, A. & Chan, W. Assessing the effect of surface chemistry on gold nanorod uptake, toxicity, and gene expression in mammalian cells. Small (Weinheim an der Bergstrasse, Germany) 4, 153 (2008).
42 Pastoriza-Santos, I., Perez-Juste, J. & Liz-Marzan, L. Silica-coating and hydrophobation of CTAB-stabilized gold nanorods. Chem. Mater 18, 2465-2467 (2006).
43 Rodriguez-Fernandez, J., Pastoriza-Santos, I., Perez-Juste, J., de Abajo, F. & Liz-Marzan, L. The Effect of Silica Coating on the Optical Response of Sub-micrometer Gold Spheres. (2007).
44 Wang, L., Zhao, W. & Tan, W. Bioconjugated silica nanoparticles: Development and applications. Nano Research 1, 99-115 (2008).
45 Ai, H., Fang, M., Jones, S. & Lvov, Y. Electrostatic layer-by-layer nanoassembly on biological microtemplates: platelets. Biomacromolecules 3, 560-564 (2002).
46 Takahashi, H. et al. Modification of gold nanorods using phosphatidylcholine to reduce cytotoxicity. Langmuir: the ACS journal of surfaces and colloids 22, 2 (2006).
47 Liao, H. & Hafner, J. Gold nanorod bioconjugates. Chemistry of materials 17, 4636-4641 (2005).
48 Wijaya, A. & Hamad-Schifferli, K. Ligand customization and DNA functionalization of gold nanorods via round-trip phase transfer ligand exchange. Langmuir 24, 9966-9969 (2008).
49 Wei, Q., Ji, J. & Shen, J. Synthesis of Near-infrared responsive gold nanorod/PNIPAAm core/shell nanohybrids via surface initiated ATRP for smart drug delivery. Macromolecular rapid communications 29, 645-650 (2008).
50 Wijaya, A., Schaffer, S., Pallares, I. & Hamad-Schifferli, K. Selective release of multiple DNA oligonucleotides from gold nanorods. ACS nano 3, 80 (2009).
51 Lee, S., Liu, G., Kim, F. & Lee, L. Remote optical switch for localized and selective control of gene interference. Nano letters 9, 562 (2009).
52 Wang, H. et al. In vitro and in vivo two-photon luminescence imaging of single gold nanorods. Proceedings of the National Academy of Sciences of the United States of America 102, 15752 (2005).
53 Durr, N. et al. Two-Photon Luminescence Imaging of Cancer Cells using Molecularly Targeted Gold Nanorods. Nano letters 7, 941 (2007).
54 Eghtedari, M. et al. High sensitivity of in vivo detection of gold nanorods using a laser optoacoustic imaging system. Nano letters 7, 1914 (2007).
55 Huang, X., El-Sayed, I., Qian, W. & El-Sayed, M. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. Journal of the American Chemical Society 128, 2115 (2006).
56 Huang, X., El-Sayed, I., Qian, W. & El-Sayed, M. Cancer cells assemble and align gold nanorods conjugated to antibodies to produce highly enhanced, sharp, and polarized surface Raman spectra: a potential cancer diagnostic marker. Nano letters 7, 1591 (2007).
57 Loo, C., Lowery, A., Halas, N., West, J. & Drezek, R. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano letters 5, 709 (2005).
58 Gobin, A. et al. Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy. Nano letters 7, 1929 (2007).
59 Dickerson, E. et al. Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice. Cancer letters 269, 57 (2008).
60 Ding, A. C., Y.; Wang, C.; Li, P.; Shieh, D. In HER-2 Antibody Conjugated Gold Nano Rod for in Vivo Photothermal Therapy. 8th IEEE Conference 882-885, 4 (2008).
61 Tong, L. et al. Gold nanorods mediate tumor cell death by compromising membrane integrity. Advanced materials (Deerfield Beach, Fla.) 19, 3136 (2007).
62 Gujrati, P. Unified lattice theory of homopolymers of general architecture: Dendrimers, stars, and branched polymers. Physical review letters 74, 1367 (1995).
63 Caminade, A., Laurent, R. & Majoral, J. Characterization of dendrimers. Advanced drug delivery reviews 57, 2130 (2005).
64 Lee, C. & MacKay, J. Designing dendrimers for biological applications. Nature biotechnology 23, 1517 (2005).
65 Hobson, L. & Harrison, R. Dendritic and hyperbranched polymers: advances in synthesis and applications. Current Opinion in Solid State and Materials Science 2, 683-692 (1997).
66 Jie, L., Jun, W., Tian-feng, W. & Li-xin, L. Synthesis and Performance of Dendrimer Polyamidoamine [J]. Chemical Researches 2 (2004).
67 Lee, J. et al. Convergent synthesis of symmetrical and unsymmetrical PAMAM dendrimers. Macromolecules 39, 2418-2422 (2006).
68 Kay, M., Glorioso, J. & Naldini, L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nature medicine 7, 33 (2001).
69 HAENSLER, J. & SZOKA, F. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate chemistry 4, 372-379 (1993).
70 Tang, M., Redemann, C. & Szoka Jr, F. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem 7, 703-714 (1996).
71 Paddison, P., Caudy, A. & Hannon, G. Stable suppression of gene expression by RNAi in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 99, 1443 (2002).
72 Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343 (2004).
73 Patri, A., Majoros, I. & Baker, J. Dendritic polymer macromolecular carriers for drug delivery. Current opinion in chemical biology 6, 466 (2002).
74 Patri, A., Kukowska-Latallo, J. & Baker Jr, J. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Advanced drug delivery reviews 57, 2203 (2005).
75 Milhem, O., Myles, C., McKeown, N., Attwood, D. & D'Emanuele, A. Polyamidoamine Starburst dendrimers as solubility enhancers. International journal of pharmaceutics 197, 239 (2000).
76 Malik, N., Evagorou, E. & Duncan, R. Dendrimer-platinate: a novel approach to cancer chemotherapy. Anti-cancer drugs 10, 767 (1999).
77 Kojima, C., Kono, K., Maruyama, K. & Takagishi, T. Synthesis of polyamidoamine dendrimershaving poly (ethylene glycol) grafts and their ability to encapsulate anticancer drugs. Bioconjugate Chem 11, 910-917 (2000).
78 Zhuo, R., Du, B. & Lu, Z. In vitro release of 5-fluorouracil with cyclic core dendritic polymer. Journal of controlled release: official journal of the Controlled Release Society 57, 249 (1999).
79 Majoros, I., Williams, C. & Baker Jr, J. Current dendrimer applications in cancer diagnosis and therapy. Current topics in medicinal chemistry 8, 1165 (2008).
80 Swanson, S. et al. Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement. International Journal of Nanomedicine 3, 201 (2008).
81 Pan, B. et al. Dendrimer-modified magnetic nanoparticles enhance efficiency of gene delivery system. Cancer research 67, 8156 (2007).
82 Pan, B. et al. Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems. Nanotechnology 20, 125101 (2009).
83 Radu, D. et al. A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent. Journal of the American Chemical Society 126, 13216 (2004).
84徐萍.上海交通大学硕士毕业论文,2007.
85潘碧峰.上海交通大学博士后论文,2007.
86 Wisher, A.C., Bronstein, I. & Chechik, V. Thiolated PAMAM dendrimer-coated CdSe/ZnSe nanoparticles as protein transfection agents. Chem. Commun. 21, 1637-1639 (2006).
87 Allman R, Cowburn P, Mason M. In vitro and in vivo effects of a cyclic peptide with affinity for theανβ3 integrin in human melanoma cells. European Journal of Cancer 36, 410-422(2000).
88 Ferrari, M. Cancer nanotechnology: opportunities and challenges. Nature reviews. Cancer 5, 161 (2005).
89 Roco, M. & Bainbridge, W. Societal implications of nanoscience and nanotechnology: maximizing human benefit. Journal of Nanoparticle Research 7, 1-13 (2005).
90崔大祥.肿瘤纳米诊断和治疗技术的研究现状与发展前景.中国肿瘤生物治疗杂志15, 5 (2008).
91崔大祥.纳米材料在肿瘤生物治疗中的潜在应用.中国肿瘤生物治疗杂志13, 4 (2006).
92 Watanabe, T. et al. Photo-thermal Ablation of Cancer by Using Photosensitive Nanoparticles.
93 Maeda, H., Sawa, T. & Konno, T. Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS* 1. Journal of Controlled Release 74, 47-61 (2001).
94 Modi, S., Prakash, J., Domb, A. & Kumar, N. Exploiting EPR in polymer drug conjugate delivery for tumor targeting. Current pharmaceutical design 12, 4785 (2006).
95 Adiseshaiah, P., Hall, J. & McNeil, S. Nanomaterial standards for efficacy and toxicity assessment.Wiey Interdiscip Rev Nanomed Nanobiotechnol,2, 99 (2010).
96 Fonsatti, E., Altomonte, M., Nicotra, M., Natali, P. & Maio, M. Endoglin (CD105): a powerful therapeutic target on tumor-associated angiogenetic blood vessels. Oncogene 22, 6557 (2003).
97 Duncan, R. & Izzo, L. Dendrimer biocompatibility and toxicity. Adv. Drug. Deliv. Rev. 57, 2215-2237 (2005).
1 Ferrari, M. Cancer nanotechnology: opportunities and challenges. Nature reviews. Cancer 5, 161 (2005).
2 Roco, M. & Bainbridge, W. Societal implications of nanoscience and nanotechnology: maximizing human benefit. Journal of Nanoparticle Research 7, 1-13 (2005).
3崔大祥.肿瘤纳米诊断和治疗技术的研究现状与发展前景.中国肿瘤生物治疗杂志15, 5 (2008).
4崔大祥.纳米材料在肿瘤生物治疗中的潜在应用.中国肿瘤生物治疗杂志13, 4 (2006).
5 Wang, Y. & Herron, N. Nanometer-sized semiconductor clusters: materials synthesis, quantum size effects, and photophysical properties. The Journal of Physical Chemistry 95, 525-532 (1991).
6徐萍. PAMAM树形分子纳米组装体系在基因递送载体中的应用.硕士学位论文上海交通大学(2007).
7 Ball, P. & Garwin, L. Science at the atomic scale. Nature 355, 761-766 (1992).
8 Caldeira, A. & Leggett, A. Influence of dissipation on quantum tunneling in macroscopicsystems. Physical Review Letters 46, 211-214 (1981).
9 Borrelli, N., Hall, D., Holland, H. & Smith, D. Quantum confinement effects of semiconducting microcrystallites in glass. Journal of Applied Physics 61, 5399 (1987).
10 Yokoyama, M. Drug targeting with nano-sized carrier systems. Journal of artificial organs 8, 77-84 (2005).
11 Jain, R. The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide)(PLGA) devices. Biomaterials 21, 2475 (2000).
12 Ito, A., Shinkai, M., Honda, H. & Kobayashi, T. Medical application of functionalized magnetic nanoparticles. Journal of bioscience and bioengineering 100, 1 (2005).
13 El-Sayed, I., Huang, X. & El-Sayed, M. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano letters 5, 829 (2005).
14 Smith, A., Dave, S., Nie, S., True, L. & Gao, X. Multicolor quantum dots for molecular diagnostics of cancer. Expert review of molecular diagnostics 6, 231 (2006).
15 Orive, G., Hern¨￠ndez, R., Gasc¨?n, A. & Pedraz, J. Micro and nano drug delivery systems in cancer therapy. Cancer Therapy 3, 131-138 (2005).
16 Portney, N. & Ozkan, M. Nano-oncology: drug delivery, imaging, and sensing. Analytical and bioanalytical chemistry 384, 620 (2006).
17 Nasongkla, N. et al. Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems. Nano letters 6, 2427 (2006).
18 Cui, D., Ozkan, C., Ravindran, S., Kong, Y. & Gao, H. Encapsulation of pt-labelled DNA molecules inside carbon nanotubes. Mechanics & chemistry of biosystems: MCB 1, 113 (2004).
19 Li, H. et al. Nanoparticle PCR: nanogold-assisted PCR with enhanced specificity. Angewandte Chemie (International ed. in English) 44, 5100 (2005).
20 Ao, L., Gao, F., Pan, B., He, R. & Cui, D. Fluoroimmunoassay for antigen based on fluorescence quenching signal of gold nanoparticles. Analytical chemistry 78, 1104 (2006).
21 Cui, D. et al. Real time PCR based on Fluorescent Quenching of Mercaptoacetic Acid-Modified CdTe Quantum Dots for Ultrasensitive Specific Detection of Nucleic Acids. Nano Biomedicine and Engineering 2, 69 (2010).
22 Pan, B. et al. Study on interaction between gold nanorod and bovine serum albumin. Colloids and Surfaces A: Physicochemical and Engineering Aspects 295, 217-222 (2007).
23 Pan, B. et al. End-to-end self-assembly and colorimetric characterization of gold nanorods and nanospheres via oligonucleotide hybridization. Nanotechnology 16, 1776-1780 (2005).
24 Cui, D. et al. Self-assembly of quantum dots and carbon nanotubes for ultrasensitive DNA and antigen detection. Analytical chemistry 80, 7996 (2008).
25 Huang, X., El-Sayed, I., Qian, W. & El-Sayed, M. Cancer cells assemble and align gold nanorods conjugated to antibodies to produce highly enhanced, sharp, and polarized surface Raman spectra: a potential cancer diagnostic marker. Nano letters 7, 1591 (2007).
26 Murphy, C. et al. Chemical sensing and imaging with metallic nanorods. Chemical communications (Cambridge, England), 544 (2008).
27 Krug, J., Wang, G., Emory, S. & Nie, S. Efficient Raman enhancement and intermittent light emission observed in single gold nanocrystals. J. Am. Chem. Soc 121, 9208-9214 (1999).
28 Qian, X. et al. In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nature biotechnology 26, 83 (2008).
29 Weissleder, R. & Mahmood, U. Molecular imaging. Radiology 219, 316 (2001).
30 Moffat, B. et al. A novel polyacrylamide magnetic nanoparticle contrast agent for molecular imaging using MRI. Molecular imaging: official journal of the Society for Molecular Imaging 2, 324 (2003).
31 Lee, J. et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nature medicine 13, 95 (2007).
32 Gao, X. et al. In vivo molecular and cellular imaging with quantum dots. Current opinion in biotechnology 16, 63 (2005).
33 Gao, X., Cui, Y., Levenson, R., Chung, L. & Nie, S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature biotechnology 22, 969-976 (2004).
34 Cui, D. et al. Fluorescent Magnetic Nanoprobes for in vivo Targeted Imaging and Hyperthermia Therapy of Prostate Cancer. Nano Biomedicine and Engineering 1, 94 (2009).
35 Durr, N. et al. Two-Photon Luminescence Imaging of Cancer Cells using Molecularly Targeted Gold Nanorods. Nano letters 7, 941 (2007).
36 Ekici, O. et al. Thermal analysis of gold nanorods heated with femtosecond laser pulses. Journal of Physics D: Applied Physics 41, 185501 (2008).
37 Hoy, C. et al. Miniaturized probe for femtosecond laser microsurgery and two-photon imaging. Optics express 16, 9996 (2008).
38 Huang, X., El-Sayed, I., Qian, W. & El-Sayed, M. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. Journal of the American Chemical Society 128, 2115 (2006).
39 Wei, K. et al. Interactions between microbubbles and ultrasound: in vitro and in vivo observations. Journal of the American College of Cardiology 29, 1081 (1997).
40 Rapoport, N., Gao, Z. & Kennedy, A. Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy. Journal of the National Cancer Institute 99, 1095 (2007).
41 Beer, A. & Schwaiger, M. Imaging of integrin |áv|?3 expression. Cancer and Metastasis Reviews 27, 631-644 (2008).
42 Semenza, G. Targeting HIF-1 for cancer therapy. Nature reviews. Cancer 3, 721 (2003).
43 Herschman, H. Molecular imaging: looking at problems, seeing solutions. Science (New York, NY) 302, 605 (2003).
44 Watanabe, T. et al. Photo-thermal Ablation of Cancer by Using Photosensitive Nanoparticles.
45 Hirsch, L. et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proceedings of the National Academy of Sciences of the United States of America 100, 13549 (2003).
46 Loo, C., Lowery, A., Halas, N., West, J. & Drezek, R. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano letters 5, 709 (2005).
47 Li, J. et al. In vitro cancer cell imaging and therapy using transferrin-conjugated gold nanoparticles. Cancer letters 274, 319 (2009).
48 Jordan, A., Scholz, R., Wust, P. & F hling, H. Magnetic fluid hyperthermia (MFH): Cancer treatment with AC magnetic field induced excitation of biocompatible superparamagnetic nanoparticles. Journal of Magnetism and Magnetic Materials 201, 413-419 (1999).
49 Ito, A., Shinkai, M., Honda, H. & Kobayashi, T. Heat-inducible TNF-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumor-targeted therapy.Cancer gene therapy 8, 649 (2001).
50 Boczkowski, J. & Lanone, S. Potential uses of carbon nanotubes in the medical field: how worried should patients be? Nanomedicine (London, England) 2, 407 (2007).
51 Kam, N., O'Connell, M., Wisdom, J. & Dai, H. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proceedings of the National Academy of Sciences of the United States of America 102, 11600 (2005).
52 Gannon, C. et al. Carbon nanotube-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field. Cancer 110, 2654 (2007).
53 Yinghuai, Z. et al. Substituted carborane-appended water-soluble single-wall carbon nanotubes: new approach to boron neutron capture therapy drug delivery. Journal of the American Chemical Society 127, 9875 (2005).
54 Tang, M., Redemann, C. & Szoka Jr, F. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate chemistry 7, 703.
55 Pan, B. et al. Dendrimer-modified magnetic nanoparticles enhance efficiency of gene delivery system. Cancer research 67, 8156 (2007).
56 Pan, B. et al. Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems. Nanotechnology 20, 125101 (2009).
57 Liu, M. & Fr¨|chet, J. Designing dendrimers for drug delivery. Pharmaceutical science & technology today 2, 393 (1999).
58 Kojima, C., Kono, K., Maruyama, K. & Takagishi, T. Synthesis of polyamidoamine dendrimers having poly (ethylene glycol) grafts and their ability to encapsulate anticancer drugs. Bioconjugate Chem 11, 910-917 (2000).
59 Chen, S., Zhang, X., Cheng, S., Zhuo, R. & Gu, Z. Functionalized amphiphilic hyperbranched polymers for targeted drug delivery. Biomacromolecules 9, 2578 (2008).