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Abstract

Magnetic Resonance Imaging (MRI) is meanwhile one of the most important medical diagnostic tools. Its specificity and sensitivity can be further extended by contrast agents (CAs). As many clinically valuable targets like DNA, mRNA or protein/enzymes reside inside the cell membrane, development of efficient intracellular targeted MR CA is required. However, prerequisite for intracellular targeting is not only the efficient delivery inside the cell but also the co-localization with the target. Recently, cell penetrating peptides (CPP) are used to achieve an efficient uptake of cargo molecules. However, it has been shown that these conjugates were predominantly taken up by an endosomal mechanism preventing a proper interaction with targets located in the cytosol. Cholesterol coupling has been reported to facilitate cellular import of siRNAs for effective silencing of protein expression [1]. We developed a contrast agent based on a lipid mediated delivery system by using cholesterol. Uptake and MR contrast enhancement ability was compared with a CPP based CA previously reported by our group [2]. To image the presence of specific mRNAs the probes composed of Gd-DOTA, FITC, a sequence to bind to target mRNA (DsRed), and CPP (D-Tat) or cholesterol for cellular delivery. Fmoc continuous solid phase chemistry was used for synthesis. Fluorescence and MR studies were performed using a mouse fibrosarcoma cell line expressing DsRed protein and its parent cell line deficient of the target sequence. Fluorescence spectroscopy showed that the CPP based CA (CPP-CA) could enter efficiently in both cell types without observable toxicity up to a concentration of 5μM. The cholesterol based CA (Chol-CA) was even more efficient. However, this conjugate was not soluble in aqueous solution at concentrations > 3µM. Both probes were able to enhance MRI contrast in labeled target containing as well as non-targeted parent cells. Intracellular relaxation rate increased already at a labeling concentration of 1µM for CPP-CA and 0.5µM for Chol-CA. However, fluorescence microscopy demonstrated that Chol-CA was also predominantly localized inside endosomes. Coupling of cholesterol further improved uptake and contrast enhancement. However, the reduced solubility in physiological aqueous media is restricting the applicability for MR imaging purposes. In addition, endosomal entrapment poses a still unsolved problem. Modifications to circumvent both drawbacks have to be implemented to achieve a sufficient cytosolic distribution of CA.