Size-dependant cellular uptake of dendritic polyglycerol.

To study the mechanism of cellular internalization, hyperbranched polyether derivatives consisting of amino-bearing hyperbranched polyglycerols (HPGs) of varied molecular mass and size range are designed and synthesized. HPGs were further fluorescently labelled by conjugating maleimido indocarbocyanine dye (ICC-mal). The conjugates are characterized by UV-vis spectroscopy, fluorescence profile, zeta potential, and dynamic light scattering. The uptake mechanism is 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). The HPG nanocarriers discussed here have many biomedical implications, particularly for delivering drugs to the targeted site.

Semaphorin 3F Promotes Transendothelial Migration of Leukocytes in the Inflammatory Response After Survived Cardiac Arrest.

Leukocyte transmigration through the blood vessel wall is a fundamental step of the inflammatory response and requires expression of adhesion molecule PECAM-1. Accumulating evidence implicates that semaphorin (Sema) 3F and its receptor neuropilin (NRP) 2 are central regulators in vascular biology. Herein, we assess the role of Sema3F in leukocyte migration in vitro and in vivo. To determine the impact of Sema3F on leukocyte recruitment in vivo, we used the thioglycollate-induced peritonitis model. After the induction of peritonitis, C57BL/6 mice were intraperitoneally (i.p.) injected daily with recombinant Sema3F or solvent for 3 days. Compared with solvent-treated controls, leukocyte count was increased in the peritoneal lavage of Sema3F-treated mice indicating that Sema3F promotes leukocyte extravasation into the peritoneal cavity. In line with this observation, stimulation of human endothelial cells with Sema3F enhanced the passage of peripheral blood mononuclear cells (PBMCs) through the endothelial monolayer in the transwell migration assays. Conversely, silencing of endothelial Sema3F by siRNA transfection dampened diapedesis of PBMCs through the endothelium in vitro. xMechanistically, Sema3F induced upregulation of adhesion molecule PECAM-1 in endothelial cells and in murine heart tissue shown by immunofluorescence and western blotting. The inhibition of PECAM-1 by blocking antibody HEC7 blunted Sema3F-induced leukocyte migration in transwell assays. SiRNA-based NRP2 knockdown reduced PECAM-1 expression and migration of PBMCs in Sema3F-treated endothelial cells, indicating that PECAM-1 expression and leukocyte migration in response to Sema3F depend on endothelial NRP2. To assess the regulation of Sema3F in human inflammatory disease, we collected serum samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 41). First, we demonstrated enhanced migration of PBMCs through endothelial cells exposed to the serum of patients after OHCA in comparison to the serum of patients with stable coronary artery disease or healthy volunteers. Remarkably, serum samples of OHCA patients contained significantly higher Sema3F protein levels compared with CAD patients (CAD, n = 37) and healthy volunteers (n = 11), suggesting a role of Sema3F in the pathophysiology of the inflammatory response after OHCA. Subgroup analysis revealed that elevated serum Sema3F levels after ROSC are associated with decreased survival, myocardial dysfunction, and prolonged vasopressor therapy, clinical findings that determine the outcome of post-resuscitation period after OHCA. The present study provides novel evidence that endothelial Sema3F controls leukocyte recruitment through a NRP2/PECAM-1-dependent mechanism. Sema3F serum concentrations are elevated following successful resuscitation suggesting that Sema3F might be involved in the inflammatory response after survived OHCA. Targeting the Sema3F/NRP2/PECAM-1 pathway could provide a novel approach to abolish overwhelming inflammation after resuscitation.

Receptor mediated cellular uptake of low molecular weight dendritic polyglycerols.

The development of effective polymer-based nanocarriers which are able to target diseased tissues still remains a great challenge in current research. Dendritic polyglycerols have emerged as novel polymeric scaffolds that have demonstrated a great potential for diverse biomedical applications. These architectures have already proven their usefulness in therapeutic approaches related to multivalency, given by the synergy between the nanosized dimensions combined with the high density of functional groups. However, a continuous effort is necessary to modify and tailor polyglycerol architectures to fit the future demands of biomedical applications. The present work deals with the development of a general synthetic strategy that allows the linkage of low molecular weight dendritic polyglycerols to fluorescent dyes and cell targeting ligands. The receptor mediated cellular uptake of the polyglycerol conjugates highlight their potential to acts as new targeted nanocarriers that should be able to decrease non-specific cellular uptake.