精选
纳米粒子瞄准癌转移目标
诸平
Figure 1. Illustration of (a) the dual-ligand nanoparticle and (b) targeting of the nanoparticles to metastatic sites usingvascular targeting and a dual-ligand strategy. Inset: Interactions of circulating tumor cells and vascular bed.
Figure 2. Evaluation of the ability of the dual-ligand nanoparticles to target metastasis in vivo in the MDA-MB-231 mousemodel. (a) The synopsis shows the timeline and schedule of the in vivo imaging studies. After 25 days from systemic injectionof MDA-MB-231 cells via the tail vein, bioluminescence imaging (BLI) showed the development of metastasis in the lungs.Each metastatic site was numbered, which is indicated on the BLI images. (b) Representative fluorescence moleculartomography (FMT) images of the mouse with metastatic spots 4 and 5. FMT imaging was performed 3 h after injection of acocktail of RGD-NP, PSN-NP, and dual-ligand-NP. (c) Using the different NIR fluorophores on each nanoparticle variant, thefluorescence signal in the thoracic region of the FMT images was quantified for each formulation (n = 5 mice). On the basis ofphantom measurements of each formulation using the FMT system, the fluorescence signal was converted to nanoparticleconcentration (mean ( SD; y-axis is in logarithmic scale). (d) The total number of nanoparticles for PSN-NP, RGD-NP, and dualligand-NPis shown for each metastatic spot (y-axis is in logarithmic scale).
Figure 3. Evaluation of the ability of the dual-ligand nanoparticles to target metastasis in vivo in the 4T1 mouse model. (a) Thedifferent protocols of in vivo imaging and ex vivo and histological analysis are shown. (b) BLI images show a mouse before andafter resection of the primary tumor. (c) ROIs indicate the location of the different organs in an FMT image. (d) RepresentativeFMT images of a mouse with 4T1 metastasis are shown 3 h after injection of a cocktail of RGD-NP, PSN-NP, and dual-ligand-NP.After thresholding, the fluorescence signal of each formulation was color-coded (green: RGD-NP; red: PSN-NP; blue: dualligand-NP).(e) Using a CRi Maestro fluorescence imaging system, ex vivo imaging of lungs indicated the colocalization of thetargeted nanoparticles and 4T1 metastatic cells expressing GFP. (f) Using the ex vivo images to confirm the location ofmetastatic sites in each mouse, the targeting accuracy of the RGD-NP, PSN-NP, and dual-ligand-NP was calculated as apercentage of the metastatic sites being successfully captured by each formulation (n = 7 mice; *p < 0.05 by Student's t test)
Figure 4. Spatiotemporal targeting of P-selectin and Rvβ3 integrin in vivo in the 4T1 mouse model. (a) Representative FMTimages show targeting of nanoparticles to micrometastases 1 h postinjection in a mouse bearing 4T1 breast cancermetastasis. Mice with 4T1 metastasis (n = 8 mice) were injected with a cocktail of Rvβ3 integrin-targeting nanoparticles (RGDNP),P-selectin-targeting nanoparticles (PSN-NP), and nontargeted nanoparticles (NT-NP) containing an equal number ofparticles per formulation. (b) Quantification of the fluorescence signal from hot spots in the FMT images is shown for eachformulation. Each animal presented 24 hot spots (total 22 hot spots). The animals were imaged at three different time points(t = 19, 22, and 26 days after tumor inoculation) to capture an early stage and later stages of metastatic disease. (c) Two hotspots from the same animal show the time-course of signal for RGD-NP and PSN-NP. (d) Comparison of the signal for RGD-NPand PSN-NP was performed for each hot spot in the early-stage (t = 19 days) and late-stage disease (t = 26 days). To consider asignal of a targeted nanoparticle superior than that of the other formulation, it had to exhibit a difference of 100 pmol of thefluorophore or greater, which corresponded to the considerable difference of 4 1011 nanoparticles (～10% of the injecteddose). Only signals above 80 pmol of fluorescence were included in the analysis, since this was the detection threshold for the“nonspecific” signal of the nontargeted formulation.
Figure 5. Targeting early stage metastasis using the dual-ligand nanoparticle. (a) The timeline and schedule is shown for thein vivo imaging studies and post mortem analyses. (b) Using whole-body planar gamma scintigraphy, a representative coronalimage shows the accurate targeting of a 99mTc-labeled dual-ligand-NP to early-stage metastases in the lungs of a mouse with4T1 metastasis 2 h after injection. Following a systemic injection of ～20 μCi of 99mTc-dual-ligand-NP, the animals were imagedusing a Gamma Medica X-SPECT system. The animal model was used 9 days after orthotopic tumor inoculation in a mammaryfat pad, which was the time point of early onset of lung metastasis. (c) In the end of the in vivo imaging session, the lungs of theanimals were perfused, excised, and imaged ex vivo using planar gamma scintigraphy, planar fluorescence imaging, and 3Dcryoimaging. 3D-cryoimaging provided an ultra-high-resolution fluorescence volume of the lungs showing the early onsetand topology of metastatic disease. (d) Overlaying ex vivo planar fluorescence and scintigraphy imaging of lungs, thecolocalization of dual-ligand-NP and 4T1 metastatic cells is shown. (e) The targeting efficacy of dual-ligand-NP was comparedin mice bearing 4T1 metastasis and healthy mice (n = 4). Using gamma scintigraphy, both groups of animals were imaged 2 hafter systemic injection of ～20 μCi of 99mTc-dual-ligand-NP. The signal intensity in the thoracic region was comparedquantitatively between the two groups.
据美国化学会主办的《化学与工程新闻》（C&EN）周刊网站2015年8月26日报道，美国凯斯西储大学（Case Western Reserve University）生物医学工程系、放射学系（Department of Radiology）、成像研究案例中心（Case Center for Imaging Research）以及综合性肿瘤中心（Comprehensive Cancer Center）的研究人员合作,采用双配位纳米粒子（dual-ligand nanoparticle）将两种在癌细胞内发现的转移性标记作为目标，追踪肿瘤细胞其从原发位向其他位的转移。图1就是肿瘤目标。对小鼠乳腺组织接种乳腺癌细胞之后9天内，此鼠的肿瘤发展转移到肺部。放射性标记的纳米粒子将2种早期转移突出明亮的恶性细胞标记作为目标。
Fig. 1 TARGETING TUMORS
Metastatic tumors developed in the lungs of this mouse within nine days after its mammary tissue was seeded with breast cancer cells. Radioactively labeled nanoparticles that target two markers of early-stage metastasis highlighted the malignant cells.
Credit: ACS Nano
尽管大多数癌症疗法治疗肿瘤是将其作为一个整体来进行处理,但是，细胞进化和行为改变是随时间而变化的。例如,它们可以改变其基因表达模式，以逃离原发肿瘤和扩散到全身。现在,研究者们已经开发出一种纳米微粒,在转移的2个不同阶段将肿瘤细胞作为目标,这有可能可以防止肿瘤扩散，相关研究结果于2015年8月23日已经在《美国化学会纳米》（ACS Nano）杂志网站发表——详见Elizabeth Doolittle, Pubudu M. Peiris, Gilad Doron, Amy Goldberg, Samantha Tucci, Swetha Rao, Shruti Shah, Meilyn Sylvestre, Priya Govender, Oguz Turan, Zhenghong Lee, William P. Schiemann, Efstathios Karathanasis. Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis. ACS Nano, 2015, 9(8): 8012-8021. DOI: 10.1021/acsnano.5b01552. Publication Date (Web): July 23, 2015.
良性肿瘤无转移。恶性肿瘤则很容易发生转移，其转移方式有4种: ①直接蔓延到邻近部位; ②淋巴转移:原发癌的细胞随淋巴引流，由近及远转移到各级淋巴结，也可能超级转移;或因癌阻碍顺行的淋巴引流而发生逆向转移。转移癌在淋巴结发展时，淋巴结肿大且变硬，起初尚可活动，癌侵越包膜后趋向固定，转移癌阻碍局部组织淋巴引流，可能引起皮肤、皮下或肢体的淋巴水肿; ③血行转移:癌细胞进入血管随血流转移至远隔部位如肺、肝、骨、脑等处，形成继发性肿瘤; ④种植:瘤细胞脱落后种植到另一部位，如内脏的癌播种到腹膜或胸膜上。显然，恶性肿瘤转移将增加对机体的损害作用，而且影响转归。
癌症死亡人数当中大约有90%的人并不是由于最初的肿瘤而导致死亡，而是因为次生肿瘤即肿瘤转移所致,经常在肺部、骨骼、肝脏以及大脑中扎根。凯斯西储大学生物医学工程师埃夫斯塔西奥斯·卡拉塞纳希斯（Efstathios Karathanasis）指出，这些转移性细胞一般会幸免于化疗，“在体内隐藏于大量的健康细胞之中，要彻底灭杀干净，其药物浓度会如此之高,以至于会置病人于死地。”如何实现在有效灭杀癌细胞的同时，又不会伤及健康细胞，这一直是科学家研究重点。通过在纳米微粒内包装小分子药物,有很多研究人员都希望开发出，仅仅为肿瘤细胞提供高浓度治疗剂量药物,同时有不会对健康组织造成影响的方法。埃夫斯塔西奥斯·卡拉塞纳希斯等人，他们通过一种配体来修饰承载药物的纳米微粒，将在癌细胞上发现的一种标识物作为靶标。埃夫斯塔西奥斯·卡拉塞纳希斯想进一步完善这种活动目标的方法。他说，癌症生物学建议针对一种癌症标志物是不够的。因为，来自同一个病人、同一肿瘤的所有癌细胞,在给定某一时间内，并非单一的特征蛋白质受体会出现过表达。所以，他决定以2种蛋白质标记物作为目标，这2种蛋白质标记物是肿瘤细胞经过了早期阶段的转移，具有不同阶段肿瘤细胞的特征。
为了验证这一想法,他的研究团队用配体修饰了直径为100 nm的脂质体,其目标是逃离肿瘤之后的转移性癌细胞2种表面蛋白表达。此逃离肿瘤之后的转移性癌细胞，进入血管随血流循环，再转移至远隔肿瘤原位的其他部位如肺、肝、骨、脑等处，形成继发性肿瘤，这种肿瘤细胞的转移是属于血行转移。此2种蛋白质有助于血液循环肿瘤细胞退出血流，在一个新位点安营扎寨，这样它们就可以建立一个新肿瘤即形成继发性肿瘤。有一种被称之为选择素（selectin）的蛋白质,帮助肿瘤细胞在血液中循环，开始沿着血管的内壁向前滚动。而第二种蛋白质是整合素（integrin）,它帮助这些肿瘤细胞在退出血流和滋生新的肿瘤之前，牢牢地附着在血管上。
选择素（selectin）是一类异亲型结合、Ca2+依赖的细胞粘着分子，是细胞黏附分子中的一个家族，为I型单链跨膜糖蛋白，能与特异糖基识别并结合。主要参与白细胞与血管内皮细胞之间的识别与粘着。选择素主要有血小板选择素（platelet selectin）、内皮细胞选择素（endothelial selectin）、白血球选择素（leukocyte selectin）。选择素又被称为选择蛋白或选择凝集素。
整合素（Integrin）又被称为整联蛋白，是一种介导细胞和其外环境（如细胞外基质，extracellular matrixc简称ECM）之间连接的跨膜受体。在信号转导中，整合素将ECM的化学成分与力学状态等有关信息传入细胞。因此，整合素除了穿过膜的机械作用，也参与了细胞讯息、细胞周期的调节、细胞型态以及细胞的运动。通常，受体的作用是将外环境的变化通知细胞并引起细胞反应。但整合素不仅介导由外到内的信号，也介导由内到外的细胞信号。因此整合素不但将ECM的信息传递给细胞，也将细胞的状态表达给外界，从而可以迅速和灵活地响应环境中的变化，比如血液的凝固作用等。细胞外基质是由细胞分泌到细胞外间质中的大分子物质，构成复杂的网架结构，支持并连接组织结构、调节组织的发生和细胞的生理活动。
为了能够验证纳米颗粒能否找到恶性癌细胞,埃夫斯塔西奥斯·卡拉塞纳希斯研究小组对其在2个不同的转移三阴性乳腺癌小鼠模型身上进行了测试。他们对实验鼠注射了荧光性或放射性标记的纳米微粒药剂,发现此纳米微粒追踪并击中了标记物目标。卡拉塞纳希斯说，他们发现大约有90%的微转移点，即大小在10～30 μm的癌细胞簇已被捕获。因为整合素和选择素也是炎症和心血管疾病的标志物,研究人员将需要测试的此纳米微粒的副作用。除此之外，他们还计划测试承载抗癌药物纳米微粒的识别系统。
乔治亚理工学院和埃默里大学（Georgia Tech and Emory University）生物医学工程系主任拉维·贝拉姆康德（Ravi V. Bellamkonda）对双重目标的策略印象深刻。他认为，“应该认识到肿瘤并非铁板一块,在每个肿瘤内或在每一个病人体内,可能都会有处于不同发展阶段或转移性扩散的肿瘤。”将纳米治疗设计更好的与当前对于癌症生物学的理解归并在一起可能效果更佳。更多信息请浏览原文。
原文摘要如下：Abstract
Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.
转载本文请联系原作者获取授权，同时请注明本文来自诸平科学网博客。
链接地址：https://wap.sciencenet.cn/blog-212210-916870.html?mobile=1
收藏
