Ultrasound-triggered drug delivery with acoustically active red blood cells

描述（由申请人提供）：血管成像和治疗是心血管疾病管理的重要工具。超声可以成像和非侵入性地提供治疗。微泡是血管内超声造影剂，其提供真实的时间的血池成像和具有超声激活的靶向（分子）成像或药物递送载体。微泡具有短的半衰期;治疗有效载荷小。红细胞已经装载了药物并重新注射到血液中，包括成功的临床试验。它们循环数周，具有高的治疗有效载荷，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输送载体-声激活红细胞（AARBCs）-结合寿命，药物负载和声激活红细胞的靶向性。AARBC被构建为RBC，内部具有气体前体全氟化碳（PFC）液体纳米滴。捕获的PFC吸收声能，转化为气体，触发药物从RBC释放。颗粒表面是天然的;早期AARBC显示小鼠中的超声造影剂半衰期约为15分钟，比微泡长。具体目标。目标1。证明并优化声学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，以最大限度地提高PFC截留和声学触发。将在临床相关超声条件下评估AARBC的声学反应。将对小鼠施用AARBC以表征造影剂寿命、生物分布和生物相容性。目标2。开发AARBC的触发式药物输送工具。AARBC将装载钙黄绿素、多柔比星或多西环素。内容物释放和药物递送将在超声后通过荧光显微镜和通过上清液荧光进行研究。将在细胞培养药物递送模型和鼠脉管系统中研究治疗功效。目标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PEG系链连接至AARBC。流动室实验将验证AARBC与VCAM-1的结合。靶向VCAM-1的AARBC将通过在高胆固醇饮食的ApoE -/-小鼠中进行对比超声成像来评估，随后是超声触发的图像引导的AARBC内容物释放，以及血管壁中的荧光药物（多西环素）检测。

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.