Photonanomedicine against EpCAM and Ki-67 positive head and neck tumors

头颈部肿瘤是一种发病率呈上升趋势的恶性肿瘤，到目前为止，手术切除是主要的治疗方案，它与面部畸形有关，复发的风险很高。因此，开发治疗该类型肿瘤的新疗法具有很高的临床价值。我们计划使用光肿瘤医学作为基础来开发这种方法，并在动物实验中对其进行评估。在之前的项目中，我们成功地开发了针对Ki-67阳性细胞的靶向概念工具箱，该工具箱基于纳米结构和光照射。制备了抗Ki-67抗体Tubb-9与不同光敏剂的抗体结合物，并对其进行了体内检测。偶联到不同的细胞和细胞核，穿透肽或包裹到脂质体中，确保细胞和核的传递。通过光化学内化(PCI)从内体释放抗体的细胞内大大提高了效率。我们能够证明以下几点，这为新方法的有效性提供了强有力的证据：1.抗体-染料结合物在组织中的可靠分布以及脂质体和光化学内化(PCI)在细胞中的摄取。通过靶向肿瘤特异性膜蛋白EpCAM和局部光照，具有明显的肿瘤选择性。一种导致细胞死亡的有效机制。这是由Ki-67的光灭活确保的，这在抗体浓度在纳摩尔范围内是可行的。在拟议的项目中，抗体辅助的光医学疗法将被简化，并在头颈部鳞状细胞癌的小鼠模型中进行测试。为了提高肿瘤的选择性，将使用针对EpCAM蛋白的脂质体。EpCAM不仅被用作脂质体运输的目标结构，而且它也将被作为光灭活的直接目标进行研究。对于肿瘤小鼠模型，将使用GFP转基因细胞，这将使荧光辅助识别肿瘤细胞和通过活体多光子显微镜在活体小鼠中进行机制研究成为可能。对于光化学内化，将使用已经在临床研究中使用的光敏剂Amphinex。在细胞穿透性免疫结合物的帮助下，辐射后增殖的细胞将被有效地破坏。将评估动物模型的短期和长期治疗成功，并将在多光子显微镜的帮助下研究Ki-67失活的细胞效应。在动物模型中成功消除肿瘤将是将光疗法应用于人类头颈部肿瘤治疗的重要第一步，为这种严重疾病提供新的治疗选择。

Head and neck tumor is a malignancy with rising incidence and to date, surgical resection is the primary treatment option, which is associated with facial disfigurement and a high risk for recurrence. Therefore, the development of new therapies for this tumor type is of high clinical value. We plan to use photonanomedicine as a basis to develop such an approach and to evaluate it in animal experiments. In a previous project, we successfully developed the toolbox for a targeting concept against Ki-67 positive cells, which is based on nano-constructs and light irradiation. Antibody conjugates of TuBB-9 (an anti-Ki-67 antibody) with different photosensitizers suitable for in vivo applications were prepared and tested. Coupling to different cell and nucleus penetrating peptides or encapsulation into liposomes ensures cellular und nuclear delivery. Intracellular release of the antibodies from endososomes by photochemical internalization (PCI) drastically increased efficiency. We were able to demonstrate the following points, which provide strong evidence for the effectiveness of the new method:1. Reliable distribution of the antibody-dye conjugates in tissues and uptake in cells by liposomes and photochemical internalization (PCI).2. A pronounced tumor selectivity by targeting of the tumor specific membrane protein EpCAM and by local light application.3. An effective mechanism that leads to cell death. This is ensured by the light inactivation of Ki-67, which is feasible with antibody concentration in the nanomolar range.In the proposed project, the antibody-assisted photomedical therapy shall be simplified and tested in a mouse model for head and neck squamous cell carcinoma. To increase the tumor selectivity, liposomes directed against the EpCAM protein will be used. EpCAM is not only used as a target structure for the transport of liposomes, but it will also be investigated as a direct target for light inactivation. For the tumor mouse model, GFP transfected cells will be used, which enable fluorescence assisted identification of tumor cells and mechanistic studies in living mice by intra-vital multiphoton microscopy. For photochemical internalization, the photosensitizer Amphinex, which is already used in clinical studies, will be applied. With the help of cell-penetrating immunoconjugates, proliferating cells will be effectively damaged after irradiation. Short-term and long-term treatment successes in the animal model will be evaluated and cellular effects of Ki-67 inactivation will be investigated with the help of multiphoton microscopy.A successful elimination of the tumor in the animal model would be an important first step towards the application of photonanotherapy for the treatment of head and neck tumors in humans, providing new treatment options for this crippling disease.