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: 8613265
• 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/8613265
• 关键词: Address; Antibodies; BODIPY; Cells; Color; Detection; Development; Energy Transfer; Epidermal Growth Factor; Excision; Exhibits; Family; Fluorescence; Fluorescent Probes; Generations; 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; public health relevance; 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）：拟议研究的目的是通过新一代荧光探针提高肿瘤可视化的选择性和敏感性，从而改善荧光引导手术中的肿瘤切除。迫切需要一种方法，帮助在手术中完全切除肿瘤病灶，同时尽量减少健康组织的切除。荧光实时肿瘤可视化能够确定肿瘤边缘，并允许检测亚毫米肿瘤，这是不可见的白光。因此，与标准手术相比，荧光引导手术降低了癌症复发的可能性，提高了患者的生存率。然而，用于肿瘤可视化的荧光探针有几个不理想的特性：(1)大多数探针提供低肿瘤-背景荧光比；(2)多种肿瘤标志物的同时靶向和可视化极为困难；(3)不可能区分位于表面和深层组织的肿瘤。在这项工作中，我们将通过荧光探针的发展来解决这些缺点，以进一步提高小尺寸肿瘤的检测和肿瘤与健康组织的区分。开发这种探针的关键障碍是缺乏合适的荧光团。现有荧光团的光学特性使得同时靶向多个肿瘤标记物非常困难。此外，目前可用的分子荧光探针不能提供确定肿瘤在组织中定位深度的能力。最近，我们已经证明了一种细菌氯-半乳糖化人血清白蛋白偶联物在体内腹腔卵巢癌转移中具有很高的选择性和灵敏度。该探针的高选择性和灵敏度是由于细菌氯荧光在附着于蛋白质时猝灭，荧光激活仅在靶细胞中发生。此外，我们已经证明，细菌氯能够区分位于表面和深层组织的肿瘤，因为它能够被绿光和近红外光激发。在