Novel Activatable Fluorophores for Multicolor Fluorescence-Guided Cancer Surgery

描述(申请人提供)：拟议研究的目标是通过新一代荧光探针提高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。迫切需要在手术中帮助完全切除肿瘤病变的方法，同时将健康组织的切除降至最低。荧光实时肿瘤可视化能够确定肿瘤边缘，并能够检测到白光看不见的亚毫米级肿瘤。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，提高了患者的存活率。然而，用于肿瘤可视化的荧光探针具有一些不理想的特性：(1)大多数探针提供较低的肿瘤与背景荧光比率；(2)同时靶向和可视化多个癌症标记物是极其困难的；以及(3)无法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，以进一步改进小尺寸肿瘤的检测和肿瘤与健康组织之间的区分。开发这类探针的关键障碍是缺乏合适的荧光团。可用的荧光团的光学性质使得同时针对多个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能确定肿瘤在组织中的定位深度。最近，我们已经证明了细菌氯-半乳糖标记的人血清白蛋白结合物在体内显示腹膜卵巢癌转移的高选择性和高敏感性。该探针的高选择性和高灵敏度源于细菌素与蛋白质结合时荧光的猝灭，以及仅在靶细胞中发生的荧光激活。此外，我们已经证明，细菌素能够区分位于表面和深层组织中的肿瘤，因为它能够被绿光和近红外光激发。在……里面 此外，细菌素表现出极窄的发射波段，通过简单的结构改造，波长可在近红外区域(700-800 nm)内调节，使其非常适合于多颜色同时检测多个目标。在这里，我们建议开发一系列具有荧光的细菌素衍生物，这些衍生物优先在靶细胞中被激活。这些衍生物将拥有共同的绿色和近红外激发波长和独特的、分辨率良好的发射波长，从而能够选择性地显示位于表面和深层组织中的肿瘤，并同时瞄准多个标记物。随后，优化后的荧光团将与靶向蛋白偶联，所产生的探针在多色荧光引导手术中的性能将在体内确定。将实现以下具体目标：(1)开发具有靶标特异性可激活荧光的细菌素家族。一类具有700-800 nm发射带的细菌素衍生物将被开发出来，在与模型蛋白质的结合时具有很高的荧光猝灭比率，在蛋白酶诱导的蛋白质降解时具有较高的去猝灭比率。(2)开发用于同时靶向多个标记物的荧光团家族。为了将目标1中开发的细菌素用于多色荧光引导手术，将开发一系列它们的衍生物，在可见光(500 Nm)和近红外(~675)区域具有共同的激发波长，以及独特的发射波长。将合成一系列基于细菌氯的能量转移阵列，以及在500 nm(BODIPY)和675 nm(氯离子)处强吸收的附加发色团。合适的亲水衍生物将被开发用于与蛋白质的结合。(3)卵巢癌腹膜转移多色荧光探针的研制与评价。在AIM 2中开发的两个独特的荧光团将分别连接到半乳糖化的人血清白蛋白和曲妥珠单抗。所得结合物将用于对过度表达凝集素和表皮生长因子的卵巢癌细胞进行双色检测。将比较双色和单色检测的灵敏度和特异度。

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.