Phospholipase-activated theranostics for PDT of breast cancer

在该提案中，我们开发了双重成像和靶向光敏剂，这些光敏剂专门设计为磷脂酰胆碱特异性磷脂酶C（PC-PLC）的底物。PC-PLC在乳腺癌中过表达且高度活跃，并且存在于质膜的外小叶上。在具有高增殖潜力的侵袭性治疗抗性三阴性乳腺癌细胞中检测到最高水平的PC-PLC表达。PC-PLC在癌细胞中的选择性区室化和同时激活表明，这种代谢酶可能用作诊断和治疗乳腺癌的靶标，包括具有高转移潜能的三阴性型。最近，我们成功地设计、合成并测试了一种近红外荧光自猝灭磷脂探针，该探针对PC-PLC具有高灵敏度和选择性。我们的探针在天然状态下是暗的，但在通过PC-PLC水解后变成荧光的。该探针中所含的荧光团焦脱镁叶绿酸a也可以作为光敏剂，因此该探针可以作为成像和光动力治疗（PDT）剂。因此，本提案的目标是使用PC-PLC作为乳腺癌检测和PDT治疗的新靶点。由于PC-PLC探针是高度亲脂性的，我们已经开发了基于脂质的纳米颗粒（LNP）以将探针递送到体内肿瘤部位。LNP的组成将被优化以制造具有降低的非特异性吸附和高探针有效载荷的小尺寸颗粒。当探针插入癌细胞膜时，PC-PLC将诱导特异性切割，恢复荧光和光毒性。我们将采用一组人乳腺癌细胞系，并检查PC-PLC探针摄取和激活，探针诱导的酶内化，确定亚细胞探针定位和测量光毒性。我们将比较乳腺癌细胞系（包括三阴性细胞）与正常组织细胞中PC-PLC活性的水平。这一差异将为体内PDT过程中乳腺癌细胞的成像和选择性损伤提供基础。将人乳腺癌细胞移植到无胸腺裸鼠中以研究我们的PC-PLC探针在体内的成像潜力。我们将测量探针递送到肿瘤部位、探针激活的动力学和灵敏度以及探针生物分布。我们将采用免疫活性的同系乳腺癌小鼠模型来比较我们的PC-PLC靶向探针与非靶向探针对具有高转移潜力的乳腺肿瘤的进展的光毒性作用。将在这些肿瘤中离体评价PC-PLC表达的异质性。由于激活的探针选择性地积聚在乳房中 光动力疗法可诱导肿瘤特异性免疫反应，减少肿瘤复发和转移。该项目的完成将提供关于不同乳腺癌细胞中PC-PLC异常表达和活性的分子肿瘤学信息，以及关于PC-PLC探针作为体内成像和PDT治疗诊断剂的潜力的临床肿瘤学信息。

In the proposal we develop dual imaging and targeting photosensitizing agents that are specifically designed as substrates for phosphatidylcholine-specific phospholipase C (PC-PLC). PC-PLC is overexpressed and highly active in breast cancer and is present on the outer leaflet of the plasma membrane. The highest level of PC-PLC expression has been detected in aggressive treatment-resistant triple negative breast cancer cells with high proliferative potential. The selective compartmentalization and simultaneous activation of PC-PLC in cancer cells suggest that this metabolic enzyme may be useful as a target for diagnosis and treatment of breast cancer, including triple negative type with high metastatic potential. Recently, we have successfully designed, synthesized and tested a near infrared fluorescent self-quenched phospholipid probe that displays high sensitivity and selectivity to PC-PLC. Our probe is dark in the native state, but becomes fluorescent after hydrolysis by PC-PLC. The fluorophore contained in this probe, pyropheophorbide a, can also act as a photosensitizer and thus the probe can function as both an imaging and photodynamic therapy (PDT) agent. Thus the goal of this proposal is to use PC-PLC as a novel target for detection and PDT therapy of breast cancer. Since PC-PLC probes are highly lipophilic, we have developed lipid-based nanoparticles (LNPs) to deliver the probes to the tumor site in vivo. The composition of the LNPs will be optimized to fabricate small-sized particles with reduced non-specific adsorption and high probe payload. When the probes intercalate into the cancer cell membrane, PC-PLC will induce specific cleavage, restoring fluorescence and phototoxicity. We will employ a panel of human breast cancer cell lines and examine PC-PLC probe uptake and activation, the probe induced enzyme internalization, determine subcellular probe localization and measure phototoxicity. We will compare the level of PC-PLC activity in breast cancer cell lines, including triple negative cells, with cells from normal tissue. This difference will provide the basis for imaging and selective damage of breast cancer cells during PDT in vivo. Human breast cancer cells will be engrafted in athymic nude mice to study the imaging potential of our PC-PLC probe in vivo. We will measure probe delivery to the tumor site, kinetics and sensitivity of probe activation and probe biodistribution. We will employ an immunocompetent syngeneic breast cancer mouse model to compare the phototoxic effect of our PC-PLC targeted probes with untargeted probe on the progression of breast tumors with high metastatic potential. The heterogeneity of PC-PLC expression will be evaluated on these tumors ex vivo. Since the activated probe selectively accumulates in the breast cancer cell plasma membrane, PDT could induce a tumor-specific immune response, reducing tumor recurrence and metastasis. The completion of this project will provide molecular oncology information regarding abnormal expression and activity of PC-PLC in different breast cancer cells as well as clinical oncology information about the potential of PC-PLC probes as in vivo imaging and PDT theranostic agents.