Targeted Near-Infrared Probes for Image-Guided Cancer Interventions

描述（由申请人提供）：我们将通过设计、创建和测试纳米级成像探针来开发用于近红外（NIR）图像引导干预的工具。这项拟议的努力将通过三个目标来实现。目的1：设计、制造和表征一套独特的用于肿瘤细胞成像的纳米级近红外探针。目标2：实现纳米探针的目标导向组件。目标3：使用近红外显微镜生成肿瘤细胞的基于图像的空间和时间图。目标1将包括产生在850至2200 nm的波长范围内发射的水溶性NIR量子点（QD）。这将通过使用油酸在有机溶剂中稳定的市售胶体硫化铅（PbS）QD的改性来实现。油酸将被特定的硫醇化分子取代，从而使QD疏水。接下来，将使用两亲性嵌段共聚物来封装QD，并且通过部分交联外部嵌段来锁定整体结构。将通过电子显微镜以及吸光度和荧光光谱法进行表征。通过目标2，水溶性QD然后将通过与单链片段可变抗体（A33 scFv）缀合而被进一步修饰，所述单链片段可变抗体识别在95%的原发性和转移性结肠直肠癌中表达的A33表面蛋白。一旦解决了QD封端化学，将优化共价缀合方法。A33 scFv-QD缀合物的特异性和灵敏度将通过Aim 3使用人结肠癌细胞系SW 1222进行研究，对照实验使用A33非表达人结肠癌细胞系HT 29。细胞将在玻璃底培养皿中生长，随后与各种浓度的ScFv-QD缀合物一起孵育。在不同孵育时间后，洗涤细胞，并使用配备有CCD相机（明场成像）和NIR检测器的Olymus-BX 51荧光显微镜进行可视化。将收集明场和NIR图像的叠加图片，NIR图像分析将允许对相应CT抗原进行高分辨率成像，从而允许其随时间的精确定位和定位。目前的图像引导工具主要依赖于可见光光谱，这受到其穿透深度的限制。NIR探头将延长穿透深度，为外科医生提供更好的引导标记。我们的努力与国立生物医学成像和生物工程研究所的目标一致，通过整合物理，工程和生命科学来加速生物医学技术的应用。这项拟议的研究也与国家癌症研究所在推进治疗方面的使命保持一致，其目标是通过改善早期检测和诊断来预防癌症。这项初步工作将为进一步开发工具提供技术基础，这些工具不仅有助于临床医生诊断，更重要的是采用图像引导治疗。

DESCRIPTION (provided by applicant): We will develop tools for near-infrared (NIR) image-guided interventions through the design, creation and testing of nanoscale imaging probes. This proposed effort will be accomplished through three aims. Aim 1: the design, fabrication and characterization of a unique set of nanoscale NIR probes for imaging tumor cells. Aim 2: the implementation of components for target directing of nanoscale probes. Aim 3: the generation of spatial and temporal image-based maps of tumor cells using NIR microscopy. Aim 1 will comprise the creation of water-soluble NIR quantum dots (QDs) that emit in the wavelength range from 850 to 2200 nm. This will be accomplished through modification of commercially available colloidal lead-sulfide (PbS) QDs which are stabilized in organic solvents using oleic acid. Oleic acid will be replaced with specific thiolated molecules rendering the QDs hydrophobic. Next, amphiphilic block copolymers will be used to encapsulate QDs and the overall structure will be locked by partially cross-linking the outer block. Characterization will be achieved through electron microscopy together with absorbance and fluorescence spectroscopy. Through Aim 2, the water soluble QDs will then be further modified through conjugation to a single chain fragment variable antibody (A33scFv) that recognizes the A33 surface protein which is expressed in 95% of primary and metastatic colorectal cancers. A covalent conjugation method will be optimized once the QD capping chemistry has been resolved. The specificity and sensitivity of the A33scFv-QD conjugates will be investigated through Aim 3 using the human colon carcinoma cell line SW1222 with control experiments using the A33-nonexpressing human colon cancer cell line HT29. The cells will be grown in glass-bottom Petri dishes and subsequently incubated with various concentrations of ScFv-QD conjugates. After various incubation times, cells will be washed and visualized under using an Olymus-BX51 fluorescence microscope equipped with both a CCD camera (bright-field imaging) and a NIR detector. Overlaid pictures of bright field and NIR images will be collected and NIR image analysis will permit high resolution imaging of the respective CT antigens allowing for their precise positioning and localization over time. The current sets of image-guided tools largely depend on the visible light spectrum which is limited by its depth of penetration. NIR probes will extend penetration depths providing better markers for guiding the surgeon. Our efforts are in line with the goals of the National Institute of Biomedical Imaging and Bioengineering in accelerating the application of biomedical technologies through integrating the physical, engineering, and life sciences. The proposed study is also aligned with the mission of the National Cancer Institute in advancing therapeutic delivery and it's goal to preempt cancer through improvements in early detection and diagnostics. This initial work will provide the technical foundation for the further development of tools that will help clinicians not only diagnose but more importantly employ image-guided therapies.