Limited utility of acetoxymethyl (AM)-based intracellular delivery systems, in vivo:: interference by extracellular esterases
Limited utility of acetoxymethyl (AM)-based intracellular delivery systems, in vivo:: interference by extracellular esterases
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DOI：
10.1111/j.1365-2818.2007.01755.x
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发表时间：
2007-04-01
影响因子：
2
通讯作者：
Balaban, Robert S.
中科院分区：
文献类型：
--
作者：
Jobsis, Paul D.;Rothstein, Emily C.;Balaban, Robert S.
使用乙酰氧基甲基（AM）基团在细胞内递送和捕获外源性光学探针是细胞生物学/生理学中的既定工具，然而，这些探针尚未在体内广泛使用。在这项研究中，使用的乙酰氧基甲基光学探针输送系统进行了评价，在体内。最初的研究表明，即使在活体动物中注射接近饱和水平的探针，在完整组织中也很少捕获探针。我们测试的假设，细胞外酯酶迅速切割乙酰氧基甲基阻止探针进入细胞，在体内。11乙酰氧基甲基探针在稀释的猪血浆中的水解速率揭示了基本上一级高速率染料裂解，其半衰期在分钟或更少的数量级上。这些血浆研究表明，乙酰氧基甲基探针在有机会进入体内组织中的细胞之前被切割。这一点通过在肌肉组织中使用活体2-光子激发显微镜得到证实，其中发现几种乙酰氧基甲基探针在输注期间在血管空间中快速裂解，而不会被捕获在肌肉细胞中。用琥珀酰亚胺酯进行的研究表明，琥珀酰亚胺酯在切割时应迅速结合蛋白质，但在体内也未能进入细胞，这与切割发生在细胞外空间的观点一致。这些数据表明，高水平的血浆和细胞外酯酶活性使经典的乙酰氧基甲基探针监测细胞内事件，在体内无效。不同的方法来捕获外源性探针将需要探索使用活体显微镜的生理研究。
The use of acetoxymethyl (AM) groups to deliver and trap exogenous optical probes inside cells is an established tool in cell biology/physiology, however, these probes have not been used extensively in vivo. In this study, the use of the acetoxymethyl delivery system for optical probes was evaluated, in vivo. Initial studies revealed very little trapped probe in intact tissues even when near saturating levels of probe were injected in living animals. We tested the hypothesis that extracellular esterases rapidly cleave the acetoxymethyl groups preventing the probes from entering cells, in vivo. The rates of hydrolysis of 11 acetoxymethyl probes in diluted porcine plasma revealed an essentially first order high rate dye cleavage with half times on the order of minutes or less. Studies on mice and rabbits revealed rates 10- to 2-fold higher, respectively. These plasma studies suggested that the acetoxymethyl probes were being cleaved before having a chance to enter cells in tissues in vivo. This was confirmed using intravital 2-photon excitation microscopy in muscle tissue where several acetoxymethyl probes were found to rapidly cleave in the vascular space during infusion and not be trapped in the muscle cells. Studies with succinimidyl esters that should quickly bind to proteins on cleavage also failed to enter cells, in vivo, consistent with the notion that the cleavage was occurring in the extracellular space. These data suggest that the high level of plasma and extracellular esterase activity render the classical acetoxymethyl probes ineffective for monitoring intracellular events, in vivo. Different approaches to trapping exogenous probes will need to be explored for physiological studies using intravital microscopy.