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近红外(NIR)图像引导干预工具。这项拟议的努力将通过三个目标来实现。目的1：设计、制作和表征一套独特的用于肿瘤细胞成像的纳米近红外探针。目标2：纳米级探针靶向组件的实现。目的3：利用近红外显微镜生成基于图像的肿瘤细胞空间和时间图谱。目标1将包括创建在850至2200 nm的波长范围内发射的水溶性近红外量子点(QD)。这将通过修饰商业上可获得的胶体硫化铅(PBS)量子点来实现，这些量子点在有机溶剂中使用油酸稳定。油酸将被特定的硫醇化分子取代，使量子点变得疏水。接下来，将使用两亲性嵌段共聚物来包裹量子点，并通过部分交联外层嵌段来锁定整体结构。表征将通过电子显微镜、吸光度和荧光光谱来实现。通过AIM 2，水溶性量子点将通过连接到识别A33表面蛋白的单链片段可变抗体(A33scFv)进行进一步修饰，A33表面蛋白在95%的原发和转移性结直肠癌中表达。一旦QD封端化学得到解决，共价共轭方法将被优化。A33scFv-QD结合物的特异性和敏感性将通过AIM 3使用人结肠癌细胞系SW1222进行研究，并使用不表达A33的人结肠癌细胞系HT29进行对照实验。细胞将在玻璃底培养皿中培养，然后与不同浓度的单链抗体-QD偶联物孵育。在不同的孵育时间后，细胞将被清洗并在奥林巴斯-BX51荧光显微镜下可视化，该显微镜同时配备了CCD摄像头(明场成像)和近红外探测器。将收集亮场和近红外图像的重叠图像，近红外图像分析将允许对各自的CT抗原进行高分辨率成像，从而允许它们随着时间的推移进行精确定位和定位。目前的成套图像引导工具在很大程度上依赖于可见光光谱，而可见光光谱受到穿透深度的限制。近红外探头将延长穿透深度，为指导外科医生提供更好的标记。我们的努力与国家生物医学成像和生物工程研究所的目标一致，即通过整合物理、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提供方面的使命保持一致，其目标是通过改进早期发现和诊断来抢占癌症的先机。这项初步工作将为进一步开发工具提供技术基础，这些工具将不仅帮助临床医生诊断，更重要的是使用图像引导疗法。

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.