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

头颈部肿瘤是一种发病率不断上升的恶性肿瘤，迄今为止，手术切除是主要的治疗选择，这与面部毁容和高复发风险有关。因此，开发新的治疗方法对这种肿瘤类型具有很高的临床价值。我们计划以光子医学为基础开发这种方法，并在动物实验中进行评估。在之前的一个项目中，我们成功地开发了一个工具箱，用于靶向Ki-67阳性细胞的概念，该工具箱基于纳米结构和光照射。制备了适合于体内应用的抗ki -67抗体TuBB-9与不同光敏剂的抗体偶联物。偶联到不同的细胞和核穿透肽或包封到脂质体确保细胞和核的递送。通过光化学内化（PCI），细胞内释放抗体的效率大大提高。我们能够证明以下几点，为新方法的有效性提供了有力的证据：抗体-染料偶联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和局部光应用具有明显的肿瘤选择性。导致细胞死亡的有效机制。这是通过Ki-67的光失活来保证的，抗体浓度在纳摩尔范围内是可行的。在本项目中，将抗体辅助光电疗法进行简化，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靶结构，而且还将被研究作为光失活的直接靶。对于肿瘤小鼠模型，将使用转染GFP的细胞，这使得荧光辅助识别肿瘤细胞和活体小鼠的多光子显微镜机制研究成为可能。对于光化学内化，将应用已用于临床研究的光敏剂Amphinex。在细胞穿透性免疫偶联物的帮助下，辐照后增殖细胞将被有效破坏。动物模型的短期和长期治疗成功将被评估，Ki-67失活的细胞效应将在多光子显微镜的帮助下进行研究。成功消除动物模型中的肿瘤将是应用光子疗法治疗人类头颈部肿瘤的重要的第一步，为这种致残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.