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。超声波可以成像，非侵入性地提供治疗。微泡是一种血管内超声造影剂，可提供实时的血池成像和具有超声激活的靶向(分子)成像或药物输送载体。微泡的半衰期很短；治疗有效载荷很小。红细胞已经装载了药物，并重新注入血液中，包括成功的临床试验。它们循环数周，治疗有效载荷高，但声学对比差，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-声学活性红细胞(AARBCs)-结合了长寿、药物有效载荷和声学激活的红细胞的靶向性。AARBC是以RBC的形式建造的，内部装有气体前驱体全氟碳(PFC)液体纳米液滴。被包裹的PFC吸收声能，转化为气体，从而触发药物从红细胞中释放。颗粒表面是天然的；早期一代的AARBC在小鼠中显示出约15分钟的超声对比半衰期，比微泡更长。明确的目标。目的#1.证明并优化具有声学活性的红细胞作为血管造影剂，延长循环寿命。AARBC的生产将得到优化，以最大限度地提高PFC捕获率和声音触发能力。AARBC的声学反应将在临床相关的超声条件下进行评估。AARBCs将用在小鼠身上，以表征对比寿命、生物分布和生物相容性。目标2.开发AARBC的触发药物输送工具。AARBC将装载钙黄素、阿霉素或多西环素。内容物释放和药物释放将在辐照后，通过荧光显微镜和上清液荧光进行研究。治疗效果将在细胞培养药物输送模型和小鼠血管系统中进行研究。目的#3.实现载药AARBC的分子靶向。抗VCAM-1的抗体将通过非共价锚定和聚乙二醇系留在AARBC上。流室实验将验证AARBC与VCAM-1的结合。针对VCAM-1的AARBC将通过高胆固醇饮食的ApoE-/-小鼠的对比超声成像进行评估，随后通过超声触发的图像引导AARBC释放内容物，以及在血管壁检测荧光药物(强力环素)。

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.