Preparation, Relaxivity In Vitro And Cellular Uptake Of Targeting Gd³⁺ Contrast Agent With Folate-BSA As Carrier

The early diagnosis of cancer is becoming more and more important with the rapid increase of cancer cases. Magnetic resonance imaging (MRI) is one of the effective tumor diagnostic techniques in clinical use. But most of MR contrast agents (including Gd3+ contrast agents) could not specifically localize in tumor tissues, so the critical importance of the field is the development of targeted contrast agents which can localize to cancer cells through a passive or active mechanism. Among all those researches, targeted drug via the folate receptor (FR), based on the fact that folate receptor can be over-expressed on the surface of some kinds of tumor cells, but scarcely expressed on normal cells, has drawn great attentions by researchers. Based on specificity of FR expression and distinctive advantages of folate, folate receptor has become an ideal target of tumor diagnosis and therapy.Based on the theory of folate-mediated delivery and the reliability and sensitivity of MRI, we have designed and prepared a new folate-mediated targeting Gd3+ contrast agent—"Folate-BSA-(Gd-DTPA)n". First, we optimized the reacting conditions of conjugation of BSA and DTPA (Diethylene triamine pentaacetic dianhydride) anhydride. Then under the optimal condition, BSA was reacted with the activated folate; after that, the conjugates were coupled with DTPA anhydride to form Folate-BSA-DTPA; finally, these coupled compounds were chelated with GdCl3. All complexes were characterized by UV, the ratios of folate and Gd-DTPA to BSA were determined by UV and ICP-AES methods respectively.The spin-lattice relaxivity of the Gd complexes in vitro were analyzed by T1-weighted magnetic resonance images.The contrast agents were labeled with fluorescein isothiocyanate (FITC) under weak base. Using KB (FR+), HeLa (FR+) and A549 (FR-) cells as model cells, the intracelluar uptake of contrast agents was observed under inverted fluorescent microscope. Besides, intracelluar uptake of contrast agents was also detected by assay of Gd3+.As a result, the ratio of folate to BSA was about 5 in every molecular of Folate-BSA-(Gd-DTPA)n , and the relaxivity was about 6×10-3 L·mmol-1ms-1 enhanced twice compared to the small molecular Gd-DTPA, though there was no difference in relaxivity between Folate-BSA-(Gd-DTPA)n and BSA-(Gd-DTPA)n. The investigation of the specific cellular uptake showed that there was a greater fluorescence intensity when FITC-labelled Folate-BSA-(Gd-DTPA)n were incubated with KB and HeLa cells than FITC-labelled BSA-(Gd-DTPA)n, and the intracellular uptake of folate complexes in KB and HeLa cells could be blocked by free folate; but there was weak fluorescent in A549 cells incubated folate-conjugated or non-conjugated contrast agents. In the test of influent factors of KB cellular uptake incubated with FITC-labelled Folate-BSA-(Gd-DTPA)n, the result showed that the intensity of cell-associated fluorescence increased with time and concentration, and decreased with ratio of Gd-DTPA to BSA. And the result of ICP-AES assay of Gd3+ also indicated that the specific cellular uptake of FR(+) with folate-conjugated contrast agents.In all, Folate-BSA-(Gd-DTPA)n thus obtained had high relaxivity and potential for targeting FR-positive tumor.