Accepted Abstracts: J Cancer Sci Ther
Anew detection mechanism for the control of successful siRNA delivery to cells or tissue is introduced using a siRNA-based probe that is capable of inducing a specific intracellular fluorescence emission. Protoporphyrin-IX (PpIX) is a fluorescent metabolite of heme-synthesis. Every nucleated human cell requires heme for heme-containing enzymes essential for the cellular energy metabolism. The last step of heme synthesis is incorporation of iron into PpIX that takes place in the mitochondria catalyzed by the enzyme ferrochelatase (FECH). Experimentally induced knock down of FECH expression in tumor cells by RNA-interference (siRNA) leads to a dramatic accumulation of fluorescent PpIX. Meanwhile, we were able to demonstrate that PpIX-fluorescence within the tumor tissue can be induced by FECH-siRNA carried by folate-coupled liposomes or dendritic polyglycerolamine nanoparticles in conjunction with a low amount of 5-ALA. PpIX-based fluorescence is excited only if the siRNA hits its target, FECH mRNA, within the cytosol. Hence, this method allows an evaluation of siRNA delivery by the detection of the newly formed PpIX- based fluorescence e.g. by flow cytometry. Due to the omnipresence of the heme-synthesis pathway, targeted application of siRNA may provide a general means for cellular imaging and determination of the successful delivery of siRNA.This approach exhibits no relevant toxicity, because siRNA-silencing of FECH leads to an endogenous and non-toxic fluorescence by affecting the cellular heme metabolism. Specific advantages of this reporter system are the positive readout of siRNA delivery, no need for transfection of a reporter gene and no need for long and complex procedures involving transcription, translation, and posttranslational modifications of the reporter molecule itself. Carriers that are able to transport FECH siRNA in a highly efficient manner may also be suitable for other siRNA-based probes. Thus, this novel reporter systemenables the selection and optimization of carriers for siRNA transport and transfection of the target tissue.