Ultrasound-triggered drug delivery with acoustically active red blood cells

描述（申请人提供）：血管成像和治疗是心血管疾病管理的重要工具。超声可以成像和无创传递治疗。微泡是血管内超声造影剂，可提供实时血池成像和靶向（分子）成像或超声激活的药物输送载体。微气泡的半衰期很短；治疗负荷很小。红细胞已被装载药物并重新注入血液，包括成功的临床试验。它们循环数周，具有很高的治疗负荷，但声学对比度差，缺乏触发药物释放的机制。我们提出了一种新的超声触发药物递送载体——声活性红细胞（aarbc），它将红细胞的寿命、药物有效载荷和靶向性与声激活相结合。AARBC是一种含有气体前驱体全氟碳（PFC）液体纳米液滴的红细胞。被捕获的PFC吸收声能，转化为气体，从而触发红细胞释放药物。颗粒表面自然；早期代AARBC小鼠超声造影剂半衰期为15 min，比微泡长。具体的目标。目标# 1。证明和优化声活性红细胞作为血管造影剂延长循环寿命。将优化aarbc的生产，以最大化PFC捕获和声学触发。将在临床相关超声条件下评估aarbc的声反应。aarbc将被施用于小鼠，以表征对比寿命、生物分布和生物相容性。目标# 2。开发AARBC的触发药物运载工具。aarbc将装载钙黄蛋白、阿霉素或强力霉素。经超声、荧光显微镜和上清荧光检测药物释放和给药情况。治疗效果将在细胞培养药物传递模型和小鼠血管中进行研究。目标# 3。实现载药AARBC的分子靶向。抗VCAM-1的抗体通过非共价锚定和PEG系链连接到aarbc上。流室实验将验证aarbc与VCAM-1的结合。在高胆固醇饮食的ApoE -/-小鼠中，通过超声造影评估AARBC对VCAM-1的靶向性，随后超声触发图像引导AARBC释放内容物，并在血管壁检测荧光药物（强力霉素）。

DESCRIPTION (provided by applicant): Vascular imaging and therapy are important tools in management of cardiovascular diseases. Ultrasound can image and non-invasively deliver therapy. Microbubbles are intravascular ultrasound contrast agents that provide blood pool imaging in real time and targeted (molecular) imaging or drug delivery vehicles with ultrasound activation. Microbubbles have a short half-life; therapeutic payload is small. Red blood cells have been loaded with drugs and reinjected in the bloodstream, including successful clinical trials. They circulate for weeks and have high therapeutic payload, but have poor acoustic contrast and lack a mechanism to trigger drug release. We propose a new ultrasound triggered drug delivery vehicle - acoustically active RBCs (AARBCs) - combining longevity, drug payload and targetability of RBCs with acoustic activation. AARBC is built as RBC with gas precursor perfluorocarbon (PFC) liquid nanodroplet inside. Entrapped PFC absorbs acoustic energy, converts to gas, which triggers drug release from RBC. Particle surface is natural; an early-generation AARBC shows ultrasound contrast half-life in mice ~15 min, longer than microbubbles. Specific Aims. Aim #1. Demonstrate and optimize acoustically active red blood cells as vascular contrast agents with extended circulation lifetime. Production of AARBCs will be optimized to maximize PFC entrapment and acoustic triggering. The acoustic response of AARBCs will be assessed at clinically relevant ultrasound conditions. AARBCs will be administered to mice to characterize contrast longevity, biodistribution and biocompatibility. Aim #2. Develop triggered drug delivery vehicles from AARBC. AARBCs will be loaded with calcein, doxorubicin or doxycycline. Contents release and drug delivery will be investigated after insonation, by fluorescence microscopy and via supernatant fluorescence. Therapeutic efficacy will be investigated in a cell culture drug delivery model and in murine vasculature. Aim #3. Achieve molecular targeting of drug-loaded AARBC. Antibody against VCAM-1 will be attached to AARBCs by noncovalent anchoring and PEG tether. Flow chamber experiments will verify binding of AARBCs to VCAM-1. AARBC targeting to VCAM-1 will be assessed by contrast ultrasound imaging in ApoE -/- mice on high-cholesterol diet, followed by ultrasound-triggered image-guided AARBC release of contents, and a fluorescent drug (doxycycline) detection in the vessel wall.