Myocyte-specific nanoplatform-enabling photodynamic cardiac ablation

描述(申请人提供)：射频或其他能量实现的局部心肌消融是目前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导致了对心脏或心脏周围组织的不必要损害，从而严重阻碍了消融的安全性和有效性。光动力疗法(PDT)已被广泛研究用于治疗癌细胞。在PDT中，被光激活后，光敏剂传递能量 与氧分子结合并产生可导致细胞死亡的活性氧物种精巧地局限于光敏细胞，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一种心脏靶向肽(CTP)，它与心肌细胞结合，而不附着于其他心肌细胞。在初步实验中，我们已经证明，在成年大鼠心肌细胞和成纤维细胞的共同培养中，CTP-PDT-NPs在激光照射后导致心肌细胞特异性损伤。因此，我们假设，在朗宁多夫灌流的大鼠心脏中，CTP-共轭PDT-NPs(CTP-PDT-NPs)将能够获得心肌细胞特异性的消融，而成纤维细胞将幸免。利用光学标测技术，我们将确定在起搏和折返期间的脉冲传播方面，在用671 nm激光照射脑室区域后，心肌细胞特异性消融与非特异性消融相比如何。在特定的目标2中，我们将确定是否可以在整个动物中实施PDT-NPs的心肌细胞特异性消融。为此，CTP-PDT-NPs将被静脉注射到麻醉的大鼠开胸模型中。10-30分钟后，我们将照亮一小块心外膜，随后将取出心脏，并进行免疫组织学研究，采用心肌细胞和成纤维细胞特异性抗体染色。这将能够评估对心肌细胞的损伤程度，以及成纤维细胞和其他非肌细胞细胞幸免的程度。此外，在一组大鼠中，胸壁在消融后将被缝合，动物将 随访1个月。然后，心脏摘除后，将进行组织学测试，以检测炎性浸润物和纤维化，并评估成纤维细胞的增殖。结果将与接受射频消融或假手术的大鼠进行比较。为了达到这个目的，我们假设体内的肌细胞特异性消融将伴随着炎症反应的减少和成纤维细胞向肌成纤维细胞的分化，以及抑制成纤维细胞的增殖和纤维化形成。

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.