Myocyte-specific nanoplatform-enabling photodynamic cardiac ablation

描述（由申请人提供）：通过射频或其他能量实现的局部心肌消融目前是药物难治性心律失常的主要治疗方式。然而，当前消融方法的高度非特异性性质是对心脏或心周组织造成不必要损伤的原因，因此严重妨碍消融安全性和有效性。光动力疗法（PDT）已经被广泛研究用于治疗癌细胞。在PDT中，在光激活后，光敏剂转移能量， 与氧分子接触并产生活性氧，活性氧诱导细胞死亡，仅限于光敏细胞，而邻近的非光敏细胞则幸免于难。在具体目标1中，我们将使用最近开发的心脏靶向肽（CTP），其结合到肌细胞，而不附着到其他心脏细胞。在初步实验中，我们已经证明，CTP-PDT-NPs在成年大鼠心室肌细胞和成纤维细胞的共培养物中递送，在激光照射后导致肌细胞特异性损伤。因此，我们假设在分离的Langendorff灌注的大鼠心脏中，CTP缀合的PDT-NP（CTP-PDT-NP）将能够获得肌细胞特异性消融，而成纤维细胞将被保留。通过光学标测技术，我们将确定在起搏和折返期间，在用671 nm激光照射心室区域后，肌细胞特异性消融与非特异性消融在脉冲传播方面的比较。在具体目标2中，我们将确定是否可以在整个动物中实施用PDT-NP的肌细胞特异性消融。为此，将CTP-PDT-NP静脉内注射到麻醉的大鼠开胸模型中。10-30分钟后，我们将照亮心室心外膜的一个小区域，随后将取出心脏，并使用肌细胞和成纤维细胞特异性抗体染色进行免疫组织学研究。这将能够评估对肌细胞诱导的损伤程度，以及成纤维细胞和其他非肌细胞细胞幸免的程度。此外，在大鼠的亚组中，胸壁将在消融后缝合，并且动物将 随访一个月。然后，在心脏取出后，将进行组织学检测，以检测炎症浸润和纤维化，并评估成纤维细胞增殖。结果将与接受射频消融或假手术的大鼠的结果进行比较。为此，我们假设体内肌细胞特异性消融将与炎症反应减少和成纤维细胞分化为肌成纤维细胞以及成纤维细胞增殖和纤维化形成抑制相关。

DESCRIPTION (provided by applicant): Regional myocardial ablation enabled by radio-frequency or other energies is currently the main treatment modality for drug-refractory arrhythmias. However, the highly unspecific nature of current ablation methodologies is responsible for unnecessary damage to cardiac or peri-cardiac tissue, thus severely hampering ablation safety and efficacy. Photodynamic therapy (PDT) has been extensively studied for treating cancerous cells. In PDT, after activation by light, photosensitizer agents transfer energy to oxygen molecules and generate reactive oxygen species which induce cell death exquisitely confined to photosensitized cells, while adjacent non photosensitized cells are spared. In specific aim 1, we will use a recently developed cardiac targeting peptide (CTP) that binds to myocytes while it does not attach to other cardiac cells. In preliminary experiments, we have demonstrated that CTP-PDT-NPs delivered in co-cultures of adult rat ventricular myocytes and fibroblasts, led to myocytes-specific damage after laser illumination. Thus, we hypothesize that in isolated Langendorff-perfused rat hearts, CTP- conjugated-PDT-NPs (CTP-PDT-NPs) will enable obtaining myocyte-specific ablation while fibroblasts will be spared. With optical mapping techniques, we will determine how myocyte-specific ablation after illumination of a ventricular region with a 671 nm Laser, compares with non-specific ablation in terms of impulse propagation during pacing and during reentry. In specific aim 2, we will determine whether myocyte-specific ablation with PDT-NPs could be implemented in the whole animal. In this aim, CTP-PDT-NPs will be injected intravenously in an anesthetized rat open chest model. After 10-30 minutes, we will illuminate a small region of the ventricular epicardium and subsequently, the heart will be removed and an immunohistological investigation with myocyte and fibroblast cell-specific antibody staining will be conducted. This will enable to assess the degree of damage induced to myocytes, and the extent to which fibroblasts and other non-myocyte cells were spared. Also, in a subgroup of rats, the chest wall will be sutured after ablation and animals will be followed-up for one month. Then, after heart removal, histological testing will be conducted to detect inflammatory infiltrates and fibrosis and assess fibroblast proliferation. Results will be compared with those of rats that underwent radiofrequency ablation or were sham operated. In this aim, we hypothesize that myocyte-specific ablation in vivo will associate with a decreased inflammatory reaction and fibroblast differentiation into myofibroblasts, as well as a dampened fibroblast proliferation and fibrosis formation.