Novel Activatable Fluorophores for Multicolor Fluorescence-Guided Cancer Surgery

用于多色荧光引导癌症手术的新型可激活荧光团

• 批准号: 8874176
• 负责人: Marcin Ptaszek
• 金额: $ 19.64万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8874176
• 关键词: Address; Antibodies; BODIPY; Cells; Color; Detection; Development; Energy Transfer; Epidermal Growth Factor; Excision; Exhibits; Family; Fluorescence; Fluorescent Probes; Generations; Health; Human; Imagery; Lectin; Lesion; Light; Malignant Neoplasms; Malignant neoplasm of ovary; Methods; Modeling; Modification; Molecular; Neoplasm Metastasis; Operative Surgical Procedures; Optics; Patients; Peptide Hydrolases; Performance; Peritoneal; Probability; Property; Proteins; Recurrence; Research; Sensitivity and Specificity; Series; Serum Albumin; Surface; Time; Tissues; Trastuzumab; Tumor Markers; Work; bacteriochlorin; bacteriochlorin a; base; cancer cell; cancer recurrence; cancer surgery; chlorin; chromophore; color detection; fluorophore; improved; in vivo; millimeter; mortality; novel; protein degradation; success; tumor

DESCRIPTION (provided by applicant): The objective of the proposed research is to improve tumor resection in fluorescence-guided surgery by improvement of selectivity and sensitivity of tumor visualization by a novel generation of fluorescence probes. There is an urgent need for methods that aid in the complete removal of tumor lesions during surgery, while minimizing resection of healthy tissue. Fluorescent real-time tumor visualization enables determination of the tumor margin and allows detection of sub-millimeter tumors, which are invisible by white light. Consequently, fluorescence-guided surgery reduces the probability of cancer recurrence and increases patient survival, compared to standard surgery. However, fluorescent probes used for tumor visualization have several properties that are not optimal: (1) The majority of probes provide a low tumor-to-background fluorescence ratio; (2) simultaneous targeting and visualization of multiple cancer markers is extremely difficult; and (3) distinguishing between tumors located on surface and in deep tissue is not possible. In this work, we will address these drawbacks through the development of fluorescent probes for further improvement in small-size tumor detection and differentiation between tumor and healthy tissue. The critical barrier in development of such probes is the lack of the suitable fluorophores. The optical properties of available fluorophores make it extremely difficult to simultaneously target multiple tumor markers. Moreover, currently available molecular fluorescent probes do not provide the ability to determine the depth of tumor localization in the tissue. Recently, we have demonstrated that a bacteriochlorin-galactosylated human serum albumin conjugate visualizes in vivo peritoneal ovarian cancer metastases with both great selectivity and great sensitivity. The high selectivity and sensitivity of this probe result from quenching of the bacteriochlorin fluorescence upon attachment to a protein, and fluorescence activation occurring only in the target cells. Moreover, we have shown that bacteriochlorin enables differentiation between tumors located on the surface and in deep tissue because of its ability to be excited by both green and near-IR light. In addition, bacteriochlorins exhibit exceptionally narrow emission bands, with wavelength tunability across the near-IR region (700-800 nm) by simple structural modifications, making them well-suited for multicolor simultaneous detection of multiple targets. Here we propose to develop a whole family of bacteriochlorin derivatives with fluorescence that is preferentially activated in the target cells. These derivatives will possess a common green and near-IR excitation wavelengths and distinctive, well resolved emission wavelengths that enable selective visualization of tumors located on the surface and in deep tissue and simultaneous targeting of multiple markers. Subsequently, the optimized fuorophores will be conjugated with targeting proteins, and the performance of the resulting probes in multicolor fluorescence-guided surgery will be determined in vivo. The following specific aims will be realized: (1) Development of a family of bacteriochlorins with target-specific activatable fluorescence. A family of bacteriochlorin derivatives, with distinctive emission bands spanning 700-800 nm, and a high ratio of fluorescence quenching upon conjugation to model proteins, and dequenching upon protease-induced protein degradation will be developed. (2) Development of a family of fluorophores for simultaneous targeting of multiple markers. To tailor bacteriochlorins developed in aim 1 to multicolor fluorescence-guided surgery, a family of their derivatives, with common excitation wavelengths in visible (500 nm) and near-IR (~675) regions and distinctive emission wavelengths, will be developed. A series of energy-transfer, bacteriochlorin-based arrays will be synthesized, with additional chromophores that strongly absorb at 500 nm (BODIPY) and 675 nm (chlorin). Suitable hydrophilic derivatives for attachment to proteins will be developed. (3) Development and assessment of fluorescent probes for multicolor detection of peritoneal ovarian cancer metastases. Two distinctive fluorophores developed in aim 2 will be conjugated to galactosylated human serum albumin and a trastuzumab antibody, respectively. The resulting conjugates will be used for two-color detection of ovarian cancer cells that over-express both lectin and epidermal growth factor. The sensitivity and specificity of two-color vs. one-color detection will be compared.

描述（由申请人提供）：拟议的研究的目的是通过提高荧光可视化的选择性和敏感性来改善荧光引导手术中的肿瘤切除，并通过新的荧光探针产生的生成。迫切需要的方法有助于在手术过程中完全清除肿瘤病变，同时最大程度地减少健康组织的切除。荧光实时肿瘤可视化可以测定肿瘤边缘，并允许检测到少量肿瘤，这是白光看不见的。因此，与标准手术相比，荧光引导的手术降低了癌症复发的可能性并增加了患者的存活率。但是，用于可视化的荧光探针具有几种最佳的特性：（1）大多数探针提供低肿瘤与背景荧光比； （2）同时对多个癌症标记的靶向和可视化非常困难； （3）不可能区分位于表面和深层组织中的肿瘤。在这项工作中，我们将通过开发荧光探针来解决这些缺点，以进一步改善肿瘤和健康组织之间的小尺寸肿瘤检测和分化。这种探针发展的关键障碍是缺乏合适的荧光团。可用荧光团的光学特性使得同时靶向多个肿瘤标记变得极为困难。此外，目前可用的分子荧光探针不能提供确定组织中肿瘤定位深度的能力。最近，我们已经证明了细菌氯酸酯 - 半乳糖基化的人血清白蛋白共轭物可视化体内腹膜卵巢癌转移，具有很高的选择性和敏感性。该探针的高选择性和敏感性是由于附着在蛋白质附着时的细菌氯氟荧光的淬灭而产生的，并且仅在靶细胞中发生荧光激活。此外，我们已经表明，由于绿色和近红外光激发其激发，细菌氯肽可以分化位于表面和深层组织中的肿瘤。在 此外，细菌蛋白表现出异常狭窄的发射带，通过简单的结构修饰，在近红外区域（700-800 nm）之间具有波长可调性，使其非常适合对多个目标的多色同时检测。在这里，我们建议开发整个具有荧光的细菌素衍生物，在靶细胞中优先激活。这些衍生物将具有常见的绿色和近IR激发波长以及独特的，分辨出良好的发射波长，从而可以选择位于表面和深层组织中的肿瘤的选择性可视化，并同时靶向多个标记。随后，将优化的实体团与靶向蛋白结合在一起，并将在体内确定多色荧光引导手术中所得探针的性能。将实现以下特定目标：（1）开发具有靶标特异性荧光的细菌蛋白家族。具有跨700-800 nm的独特发射带的细菌氯肽衍生物家族，在结合与蛋白质建模时荧光淬灭之比很高，并且会在蛋白酶诱导的蛋白质降解上去除。 （2）开发荧光团系列，以同时靶向多个标记。为了在AIM 1中量身定制的细菌菌蛋白针对多色荧光引导手术，其衍生物的家族将开发在可见的（500 nm）和近IIR（〜675）区域的常见激发波长以及独特的发射波长。将合成一系列的能量转移，基于细菌氯氯氯氯氯氯氯氯氯氯氯氯肽的阵列，其他发色团在500 nm（BODIPY）和675 nm（氯蛋白）下强烈吸收。将开发合适的亲水性衍生物，用于附着在蛋白质上。 （3）开发和评估荧光探针的多色检测卵巢癌转移。在AIM 2中开发的两个独特的荧光团将分别与半乳糖化的人血清白蛋白和曲妥珠单抗抗体共轭。所得的结合物将用于对过表达凝集素和表皮生长因子的卵巢癌细胞进行两色检测。将比较两色与单色检测的灵敏度和特异性。