Dept. of Clinical Radiology, University Hospital Muenster, Muenster, Germany.
Clinic for Radiology, St. Franziskus Hospital Muenster, Muenster, Germany; Interdisciplinary Center for Clinical Research (IZKF), University of Muenster, Muenster, Germany.
J Control Release. 2014 Jul 28;186:32-40. doi: 10.1016/j.jconrel.2014.04.053. Epub 2014 May 9.
Small molecular imaging probes are often found to be rapidly cleared from the circulation. In order to improve signal to noise ratio (SNR) by high probe accumulation in the target tissue we intended to prolong the presence of the probes in the circulation by exploiting inherent transport mechanisms. Human serum albumin (HSA) is playing an increasingly important role as a drug carrier in clinical settings and drugs directly bound to albumin or attached to albumin binding moieties have been successfully developed for treatment approaches. To optimize the bioavailability of existing fluorescent probes, a hydrophobic affinity tag is installed, which enhances albumin binding. In a first experiment an endothelin-A receptor (ETAR) probe is modified by inserting a trivalent linker, attaching an albumin affinity tag and labeling the conjugate with the fluorescent dye Cy 5.5. The spectroscopic properties of the conjugate are examined by photometer- and fluorometer measurements in comparison to a probe without albumin binding tag. Albumin binding was proven by agarose gel electrophoresis. The affinity towards ETAR was confirmed in vitro by cell binding assays on human fibrosarcoma cells (HT-1080) and in vivo by murine xenograft imaging studies. In vitro, the modified probe retains high target binding in the absence and presence of albumin. Binding could be blocked by predosing with ETAR antagonist atrasentan, proving specificity. The in vivo examinations in comparison to the established probe showed a reduced renal elimination and a prolonged circulation of the tracer resulting in significantly higher signal intensity (SI) at the target and a higher signal-to-noise ratio (SNR) between 3h and 96 h after injection. In summary, we designed a small molecular, non-peptidic fluorescent probe which targets ETAR and reversibly binds to serum albumins. The reversible binding to albumin enhances the biological half-life of the probe substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization in molecular imaging experiments.
小分子成像探针通常在血液循环中迅速清除。为了通过在靶组织中高探针积累来提高信噪比（SNR），我们试图通过利用固有转运机制来延长探针在血液循环中的存在时间。人血清白蛋白（HSA）在临床环境中作为药物载体的作用越来越重要，并且已经成功开发了直接与白蛋白结合的药物或与白蛋白结合部分结合的药物用于治疗方法。为了优化现有荧光探针的生物利用度，安装了疏水性亲和标签，增强了白蛋白结合。在第一个实验中，通过插入三价接头、连接白蛋白亲和标签并将缀合物用荧光染料 Cy 5.5 标记，来修饰内皮素-A 受体（ETAR）探针。通过光度计和荧光计测量与没有白蛋白结合标签的探针相比，检查了缀合物的光谱性质。通过琼脂糖凝胶电泳证明了白蛋白结合。通过人纤维肉瘤细胞（HT-1080）上的细胞结合测定和体内小鼠异种移植成像研究在体外证实了对 ETAR 的亲和力。在没有和存在白蛋白的情况下，修饰后的探针保留了对靶标的高结合。通过用 ETAR 拮抗剂 atrasentan 预先给药可以阻断结合，证明了特异性。与已建立的探针相比，体内检查显示出较低的肾脏清除率和延长的示踪剂循环，导致在注射后 3 小时至 96 小时之间靶标处的信号强度（SI）显著增加，并且信号与噪声比（SNR）增加。总之，我们设计了一种小分子、非肽类荧光探针，该探针靶向 ETAR 并可逆地与血清白蛋白结合。与白蛋白的可逆结合大大增强了探针的生物半衰期，并使皮下肿瘤能够进行数天的近红外光学成像。这种将探针可逆连接到血清白蛋白的方法可以作为优化示踪剂分布的工具，以在分子成像实验中更精确地对靶标进行特征描述。
