ausblenden:
Schlagwörter:
-
Zusammenfassung:
Endocytosis is a fundamental process through which essential biomolecules otherwise unable to permeate the plasma membrane are taken up by cells. This process is mainly used to fulfill the nutritional requirements of cells. The idea of exploiting this uptake mechanism to transfer exogenous macromolecules into cells is widely used. After crossing the cellular membrane via endocytosis most macromolecules enter the so-called endo-Iysosomal degradation pathway. This still is a severe hindrance for the development of therapeutics and diagnostic tools based on intracellular targets like. DNA or oligonucleotides. Cell penetrating peptides (CPPs) seemed to be promising tools for improving intracellular delivery of cargos. However, recent studies questioned the maintenance of biological activity of specific molecules following CPP-assisted uptake. For most CPPs it has now been established that the mechanism of internalization mainly involves endocytosis/macropinocytosis, by this limiting access to targets located in the cytosol. Inspired by Crotamine, a toxin in snake venom, we developed the novel CPP CyLoP-1 that was able to pass through the cellular membrane barrier. Recently, we have shown its capability of delivering a MRI contrast agent into the cytosol. Herein we present the characterization of CyLoP-1 in comparison to other well-studied CPPs. The fluorescently labeled CPPs were assessed for cellular and cytosolic localization in 3T3 fibroblasts by fluorescence spectroscopy and microscopy. We also studied the distribution in vivo by injecting CyLoP-1 into mice followed by ex vivo observation of fluorescence in organs. CyLoP-1 required the presence of serum for solubility in medium and for cytosolic delivery. Without serum CyLoP-1 stuck to the outer membrane. In contrast, arginine-rich CPPs showed diffuse labeling in serum-free medium. Cytosolic delivery of CyLoP-1 was maintained at 4°C indicating the uptake to be independent or complementary to endocytosis. In vivo studies showed that the peptide was readily dispersed into the blood stream after ip injection and it excretion via the kidneys after 4h. Apart from the kidneys and the bladder, many other organs (liver, spleen, lungs) were fluorescent. Interestingly, CyLoP-1 showed faint labeling in the brain as well. Our study reveals that CyLoP-1, a cysteine and arginine-rich peptide, is remarkably different in comparison to other available CPPs. Its peculiar cytosolic localization makes it a likely candidate for targeted delivery of drugs and agents for molecular imaging.
