Synthesis of hyperbranched polyglycerol dye conjugates for in vitro and in vivo targeting studies (2011)

In this work hyperbranched dendritic polyglycerols with different molecular weights were synthesized and functionalized. HPGs labeled with several fluorescent dyes were investigated with regard to size-dependant cellular uptake, intracellular behavior, receptor-mediated endocytosis, and their potential as transport carriers in in vivo studies. To study the mechanism of cellular internalization, hyperbranched polyether derivatives consisting of amino-bearing hyperbranched polyglycerols (HPGs) of varied molecular mass and size range were designed and synthesized. HPGs were further fluorescently labelled by conjugating maleimido indocarbocyanine dye (ICC-mal). The conjugates were characterized by UV-Vis spectroscopy, fluorescence profile, zeta potential, and dynamic light scattering. The uptake mechanism was studied by fluorescence activated cell sorting (FACS) analysis, fluorescence spectroscopy, and confocal microscopy with human lung cancer cells A549, human epidermoid carcinoma cells A431, and human umbilical vein endothelial cells (HUVEC) cells. For the first time, the results suggest that the higher- molecular-weight HPGs (40-870 kDa) predominantly accumulate in the cytoplasm much better than their low-molecular-weight counterparts (2-20 kDa). An additional study described the specific cellular uptake and the transport mechanism in the different cell organelles with the help of selective fluorescent dyes for clathrin, early endosomes, Golgi apparatus, late endosomes, and lysosomes. It could be demonstrated that cellular uptake of HPG-ICC (208 kDa) was found to occur primarily through clathrin-mediated endocytosis and probably dynamin-dependent endocytosis pathways, but the co- localization was quite weak. Particles with a molecular weight under 40 kDa were not taken up into the tumor cells. HPGs are quickly trafficked to the lysosomes, but show increased endosomal accumulation once the lysosomal compartments become saturated. It is demonstrated that HPG was not localized with the Golgi apparatus. With using additional target functionalities the possible uptake of cells with low molecular weight HPG (10 kDa) could be investigated. Towards this direction, HPG was labeled with ICC to image cancer cells and coupled with specific peptides (RGD, LHRH) to study the endocytosis pathway in three different cancer cell lines. In conclusion, it was shown that active cellular uptake is target-dependent for the polymeric class of hyperbranched polyglycerols. Based on the results of the in vitro studies in vivo studies with different sizes of hyperbranched polyglycerols coupled with Indotricarbocyanine maleimide (ITCC-mal) and a Cy5.5 derivates, respectively were performed within collaborations. It was interesting to note that the mean fluorescence signal inside the tumors was most significant after injection with 120 kDa with approximately 10 nm in size. In summary, the high molecular weight polyglycerols examined here presents themselves as an interesting platform for next generation biomaterials. The broad range of positive characteristics, such like biocompatibility, non-toxicity, high water solubility, and the possibility of multiple functionalization of the surface, renders HPGs as an attractive molecular tool for in vivo transport systems.