国家重大科学研究计划项目“生物医学纳米材料对血细胞作用的研究”工作进展与讨论

结合多模态纳米探针进行淋巴癌等血液恶性肿瘤的在体分子成像与疗效评价研究

东南大学 顾宁 教授


1. Fe3O4@Pt纳米颗粒制备及其增强过氧化物酶活性研究

我们通过一个简单的方法制备Pt修饰的Fe3O4纳米颗粒(Fe3O4@Pt NPs),对并对其结构和形貌进行了TEM、XRD、EDS等的表征。进一步的,我们研究了这种复合颗粒的类过氧化物酶的活性。其催化过程符合米氏动力学方程。结果表明,相对于Fe3O4纳米颗粒,Pt的修饰对其催化活性有明显的增强。
Ming Ma, Jun Xie, Yu Zhang, Zhongping Chen, Ning Gu, Fe3O4@Pt nanoparticles with enhanced peroxidase-like catalytic activity, Materials Letters, 2013, 105, 36-39.


Fig.1. (a) Low- and (b) high-magnification TEM photograph of Fe3O4@Pt NPs


Fig. 2. UV-vis absorption spectra of the sodium citrate buffer (pH = 4.4) containing 0.5 M H2O2 and 0.5 mM TMB in the presence of (a) Fe3O4@Pt NPs and (b) Fe3O4 NPs with equal concentration of Fe(3 µg/mL).

2.金属纳米颗粒寡聚体等离激元耦合性质的调控

金属纳米颗粒聚集体的光学性质非常复杂,个体颗粒的材质、几何结构以及空间排列都会影响等离激元的耦合。由于金属纳米颗粒可以看作一个耦合谐振子,而量子力学的共振和干涉有着类似的数学表达,因而等离激元耦合产生了许多量子物理中常见的现象,如 FANO 共振、电磁诱导透明 (EIT)、Rabi分裂等,理解其中的物理机制,控制等离激元的耦合实现特定的功能,是等离激元光子学的一个主要任务。本文基于CDA理论提出了定性估计纳米颗粒聚集体光学散射特性的散射分解方法,可以将寡聚体等离激元的二维耦合简化为两步一维耦合,从而可以方便地预测纳米颗粒寡聚体的光学性质,为等离激元耦合的人工调控提供了有力的理论工具。
Meng Wang , Min Cao , Zhirui Guo , Ning Gu,Two-Step Decomposition of Plasmon Coupling in Plasmonic Oligomers, J. Phys. Chem. C, DOI: 10.1021/jp4004054 2013.


Figure 1. Calculated extinction spectra of a heterogeneous dimer (a) and trimer (b) under transverse and longitudinal excitations by CDA. Thecolors of the sphere indicate the type of the plasmonic particles.


Figure 2. (a) Extinction, scattering and absorption cross sections of a pentamer by dynamic CDA, the pentamer consists five silver nanoparticles (a =25 nm) with gap size g = 25 nm. (b) Decomposition and dipolar fit of scattering spectrum for the pentamer. (c) Near field distributions at three specific frequencies as marked in (a).


Figure 4. Tuning of plasmon resonance of silver heptamer. The dashed line corresponds to the scattering cross section of the heptamer with gap size of 10 nm.

3. Aurora激酶小分子抑制剂AM-005的抗肿瘤活性研究

本课题主要描述从实验室前期合成的化合物中筛选出的一个具有Aurora激酶抑制活性的小分子抑制剂AM-005,以及AM-005的抗肿瘤活性。实验结果显示,AM-005可抑制来自不同组织的肿瘤细胞的生长和增殖,并且在细胞内外均能降低Aurora激酶的催化活性,与细胞孵育仅2小时即可使Aurora激酶催化底物的磷酸化程度降低。在有丝分裂细胞中,AM-005可使细胞纺锤体和染色体发生异常,出现单极纺锤体和染色体滞后现象。细胞周期实验表明,AM-005处理后的肿瘤细胞不经过胞质分裂过程,从而细胞会出现多倍体现象。裸鼠移植瘤实验中,将AM-005以灌胃的方式给药,结果显示其抗肿瘤活性较强,与阳性药AT9283相比有显著差异,说明经过结构改造后的AM-005可能在今后的临床研究中适合于口服用药。
Ming Zheng, Youguang Zheng, Li Xie, et al. Orally Active Aurora A/B Kinase Inhibitor, AM-005, Suppresses the Growth of Human Colon Carcinoma Cells. Drug Develop Res, DOI: 10.1002/ddr.21077


图1. AM-005与肿瘤细胞孵育1、2和4h后,组蛋白H3 ser10磷酸化程度的变化。


图2. AM-005处理细胞后,有丝分裂期细胞的染色体和纺锤体发生异常。


图3.将肿瘤细胞周期同步于G1/S期的交界处后释放细胞周期, 对照组(A)的细胞经历正常的有丝分裂过程,而AM-005处理组(B)不发生胞质分裂,产生多倍体细胞。

4. Design and synthesis of novel 4-benzothiazole amino quinazolines Dasatinib derivatives as potential anti-tumor agents

Dasatinib(SprycelTM)是Bristol-Myers Squibb公司研究开发一种口服多激酶抑制剂,2006年6月获得FDA优先审批,用于对既往治疗失败或不耐受的成人慢性髓细胞白血病(CML)的所有病期患者。以Dasatinib作为先导化合物,利用计算机辅助药物设计对2-氨基苯并[d]噻唑类化合物进行定量构效关系(QSAR)研究,确定该类化合物产生激酶抑制活性的结构特征;再基于构效关系分析,运用增环原理和拼合原理等药物设计的基本方法,用喹唑啉环替代Dasatinib结构中的嘧啶环,以苯并噻唑环替换噻唑环,以其它极性取代基替换4-羟乙基哌嗪部分,并用其他2,6位连有小取代基的苯胺代替2-氯-6-甲基苯胺环,成功设计出具有4-氨基喹唑啉基苯并[d]噻唑类母核的目标化合物19个。对目标化合物进行了体外抗肿瘤活性测试,19个目标化合物中有11个能够显著抑制人结肠癌细胞株(HCT-116)和(DLD1)。
J. Cai, M. Sun, X.Q. Wu, J.Q. Chen, P. Wang, X. Zong, M. Ji, Design and synthesis of novel 4-benzothiazole amino quinazolines Dasatinib derivatives as potential anti-tumor agents, Eur J Med Chem, 63 (2013) 702-712.


J. Cai, M. Sun, X.Q. Wu, J.Q. Chen, P. Wang, X. Zong, M. Ji, Design and synthesis of novel 4-benzothiazole amino quinazolines Dasatinib derivatives as potential anti-tumor agents, Eur J Med Chem, 63 (2013) 702-712.

5. MIM蛋白抑制剂多肽的设计和生物研究

MIM蛋白(也称MTSS1)是一种重要的胞内蛋白,能与细胞膜结合,在细胞的极化、 细胞运动和内吞作用等过程中发挥调节功能,其表达异常与多种肿瘤发生相关。MIM蛋白在结构上属于BAR家族成员,两个MIM蛋白分子能够借助其N端的I-BAR区域结合成二聚体的结构,这一结构能结合细胞膜并促使膜形成伪足状突起。此外MIM蛋白还有其他一些功能,如调节肌动蛋白细胞骨架、与皮层肌动蛋白等其他蛋白相互作用等。然而MIM蛋白二聚体发挥生物活性的机制尚未阐明,且未见报道有针对性的相关抑制剂。本文根据MIM蛋白I-BAR区二聚体的结构特点,设计制备了一系列多肽,并对抑制活性进行了表征,成功发现了有效的MIM蛋白抑制剂多肽。借助抑制剂多肽对MIM蛋白二聚化功能的分子机制进行研究,发现抑制二聚化作用能够减弱MIM介导的细胞膜变化,并降低MIM引起的内吞作用增加,同时不影响MIM/皮层肌动蛋白或肌动蛋白间的相互作用。数据表明,MIM双分子二聚体是该蛋白调节膜形态的结构基础,而且该过程并不依赖于MIM蛋白对肌动蛋白细胞骨架的调节。
M. Cao, T.L. Zhan, M. Ji, X. Zhan. Dimerization is necessary for MIM-mediated membrane deformation and endocytosis. Biochem J, 2012, 446: 469-475.


图1:MIM抑制剂的设计。(A)设计的MIM多肽抑制剂结构及序列;(B,C)重组多肽的表达和纯化;(D)GFP标记的抑制剂MIM-S1在细胞中与靶蛋白Flag-MIM能够结合。


图2:活性多肽抑制MIM蛋白介导的胞吞作用和膜突起。(A)表达MIM-GFP的细胞,其内吞作用显著增加,但与MIM-S1-Myc共表达时这种增加会受到抑制。(B)抑制剂与靶蛋白过表达时,MIM诱导的丝状伪足减少。(C)抑制剂不影响MIM与cortactin与actin的结合。

6. 抗ABCG2单克隆抗体联合载紫杉醇氧化铁纳米颗粒靶向治疗多发性骨髓瘤癌干细胞的研究

迄今,多发性骨髓瘤仍是不可治愈的疾病之一,究其原因,传统的治疗和现代的各种新颖的治疗仅仅杀灭了大多数的骨髓瘤细胞而没有杀灭骨髓瘤癌干细胞,幸存的骨髓瘤癌干细胞导致了多发性骨髓瘤的复发,而骨髓瘤癌干细胞高表达耐药分子ABCG2,这与其复发密切相关。为此,我们研制了新型的载紫杉醇氧化铁纳米颗粒,并采用免疫磁珠分选的方法得到表型为CD138–CD34– 多发性骨髓瘤细胞,进一步采用增殖、克隆形成、迁移、耐药以及致瘤性试验等鉴定了CD138–CD34– 多发性骨髓瘤细胞具有癌干细胞特性,随后我们应用抗ABCG2单克隆抗体靶向ABCG2分子,阻止耐药泵将进入细胞内的药物泵出细胞外,并联合载紫杉醇氧化铁纳米颗粒从体内外观察其对多发性骨髓瘤癌干细胞的效应及其机制,结果发现抗ABCG2单克隆抗体联合载紫杉醇氧化铁纳米颗粒通过诱导细胞的凋亡发挥了抗多发性骨髓瘤癌干细胞的效应。
Yang C, Xiong F, Wang J, Dou J, Chen J, Chen D, Zhang Y, Luo S, Gu N. Anti-ABCG2 monoclonal antibody in combination with paclitaxel nanoparticles against cancer stem-like cell activity in multiple myeloma. Nanomedicine (Lond). 2013 Mar 27. In press.


Figure 1. Physicochemical properties of paclitaxel iron oxide nanoparticles. (A) The structure scheme of PTX iron oxide NPs (PTX-NPs) formed from the hydrophobic interaction between the polyoxypropylene chain and oleic acid on the surface of iron oxide NPs; (B) Transmission electronmiscroscopy images of PTX-NPs after rehydration with water; (C) the hysteresis loop of PTX-NPsdetermined by a vibrating sample magnetometer, (D) the kinetics of PTX release from PTX-NPs.Fe3O4@OA: Oleic acid-capped iron oxide; H: Magnetic field; M: Magnetization; NP: Nanoparticle;PTX: Paclitaxel.


Figure 2. Analysis of CD138–CD34–cell characteristics. 1 and 2 represent CD138–CD34– cells and non- CD138–CD34– cells, respectively. (A) ABCG2 expression in CD138–CD34– cells was higher than that of non- CD138–CD34– cells detected by Western blot and GAPDH as control. (B) 1 × 106 CD138–CD34– cells or non CD138–CD34– cells were seeded into a 96-well plate and incubated for 6 days. Cell proliferative ability of CD138–CD34– cells was higher than that of non- CD138–CD34– cells. (C) The migration ability (3.83%) in CD138–CD34– cells was higher than that of non- CD138–CD34– cells (1.25%) after a 24 h incubation period. (D) Cultured with paclitaxel (8 mg) in 96-well plate for 24 h, the cell resistant rate to paclitaxel was 84.33% in CD138–CD34– cells and 36.67% in non- CD138–CD34– cells in vitro. (E) Colony capability in soft agar media for 11 days of culture. (F) Comparison of the colony capabilities between the CD138–CD34– and non- CD138–CD34– cells. (G) Tumorigenic images of CD138–CD34– and non- CD138–CD34– cells in NOD/SCID mice on day 30 after injecting 1 × 106 cells at the lower right site and 2 × 105 cells at the top left sites of mice.*p < 0.05, **p < 0.03, ***p < 0.005. GADPH: Glyceraldehydes-3-phosphate dehydrogenase; NOD: Nonobese diabetic; SCID: Severe combined immunodeficiency.


Figure 6. Anti-ABCG2 monoclonal antibodies in combination with paclitaxel iron oxide nanoparticles significantly changed the bone mineral denisty of multiple myeloma cancer stem cells xenograft mice. (A-C) Representative clinical pictures of different treated groups on week 2 after treatment. The changes of lytic bone lesions in femurs, vertebrats and skulls in multiple myeloma cancer stem-like cell xenograft nonobese diabetic/severe combined immunodeficiency mice were detected by microcomputed tomography.The upper and the lower images show prior therapy lesions in multiple myeloma cancer stem-like cell mice. The PTX-NPs, PTX and mAb groups all ameliorated lytic bone lesions in different degrees as shown in the lower images. mAb: Monoclonal antibody; NP: Nanoparticle; PTX:Paclitaxel.