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）：拟定研究的目的是通过新一代荧光探针改善肿瘤可视化的选择性和灵敏度，从而改善荧光引导手术中的肿瘤切除。迫切需要有助于在手术期间完全去除肿瘤病变，同时使健康组织的切除最小化的方法。荧光实时肿瘤可视化能够确定肿瘤边缘，并能够检测亚毫米肿瘤，这些肿瘤在白色光下不可见。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并提高了患者的生存率。然而，用于肿瘤可视化的荧光探针具有几个不是最佳的性质：（1）大多数探针提供低的肿瘤与背景荧光比率;（2）同时靶向和可视化多种癌症标志物是极其困难的;以及（3）区分位于表面和深层组织中的肿瘤是不可能的。在这项工作中，我们将通过开发荧光探针来解决这些缺点，以进一步改善小尺寸肿瘤检测和肿瘤与健康组织之间的区分。开发此类探针的关键障碍是缺乏合适的荧光团。现有荧光团的光学性质使得同时靶向多种肿瘤标志物变得极其困难。此外，目前可用的分子荧光探针不提供确定组织中肿瘤定位深度的能力。最近，我们已经证明，细菌绿素-半乳糖基化人血清白蛋白结合物可视化在体内腹膜卵巢癌转移具有很大的选择性和很高的灵敏度。这种探针的高选择性和灵敏度是由于细菌绿素在附着到蛋白质上时荧光猝灭，并且荧光激活仅发生在靶细胞中。此外，我们已经表明，菌绿素能够区分肿瘤位于表面和深部组织，因为它的能力，被激发的绿色和近红外光。在 此外，细菌绿素表现出非常窄的发射带，通过简单的结构修饰，在近红外区域（700-800 nm）具有波长可调谐性，使其非常适合于多个目标的同时检测。在这里，我们建议开发一个完整的家庭的菌绿素衍生物的荧光，优先激活的靶细胞。这些衍生物将具有共同的绿色和近红外激发波长以及独特的、良好分辨的发射波长，其使得能够选择性地可视化位于表面上和深层组织中的肿瘤并同时靶向多种标记物。随后，优化的荧光团将与靶向蛋白缀合，并且将在体内确定所得探针在荧光引导手术中的性能。具体目标如下：（1）开发具有靶向特异性可激活荧光的菌绿素家族。一个家庭的菌绿素衍生物，具有独特的发射带跨越700-800 nm，和一个高比率的荧光猝灭后，共轭模型蛋白质，和dequenching后，蛋白酶诱导的蛋白质降解将被开发。(2)开发用于同时靶向多个标记物的荧光团家族。为了使目标1中开发的细菌绿素适合荧光引导手术，将开发一系列其衍生物，这些衍生物具有可见光（500 nm）和近红外（~675）区域的共同激发波长和独特的发射波长。将合成一系列基于能量转移的菌绿素阵列，其中含有在500 nm（BODIPY）和675 nm（二氢卟酚）处强烈吸收的额外发色团。将开发用于附着于蛋白质的合适的亲水性衍生物。(3)卵巢癌腹膜转移荧光探针的开发与评价。在目标2中开发的两种独特的荧光团将分别与半乳糖基化的人血清白蛋白和曲妥珠单抗抗体结合。所得的结合物将用于过表达凝集素和表皮生长因子的卵巢癌细胞的双色检测。将比较双色与单色检测的灵敏度和特异性。