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.

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

Poly(oligo(ethylene glycol)acrylamide) brushes by surface initiated polymerization: effect of macromonomer chain length on brush growth and protein adsorption from blood plasma.

Three hydrolytically stable polyethyleneglycol (PEG)-based N-substituted acrylamide macromonomers, methoxypolyethyleneglycol (350) acrylamide (MPEG350Am) methoxypolyethyleneglycol (750) acrylamide(MPEG750Am) and methoxypolyethyleneglycol (2000)acrylamide (MPEG2000Am) with increasing PEG chain length were synthesized. Surface-initiated aqueous atom transfer radical polymerization (ATRP) using CuCl/1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA) catalyst was utilized to generate dense polymer brushes from these monomers via an ester linker group on the surface of model polystyrene (PS) particles. The molecular weight, hydrodynamic thickness, and graft densities of the grafted polymer layers were controlled by changing the reaction parameters of monomer concentration, addition of Cu(II)Cl2, and sodium chloride. The graft densities of surface-grafted brushes decreased with increasing PEG macromonomer chain length, 350 > 750 >> 2000, under similar experimental conditions. The molecular weight of grafts increased with increase in monomer concentration, and only selected conditions produced narrow distributed polymer chains. The molecular weight of grafted polymer chains differs significantly to those formed in solution. The hydrodynamic thicknesses of the grafted polymer layers were fitted to the Daoud and Cotton model (DCM) for brush height on spherical surfaces. The results show that the size of the pendent groups on the polymer chains has a profound effect on the hydrodynamic thickness of the brush for a given degree of polymerization. The new PEG-based surfaces show good protection against nonspecific protein adsorption from blood plasma compared to the bare surface. Protein adsorption decreased with increasing surface density of grafted polymer chains. Poly(MPEG750Am) brushes were more effective in preventing protein adsorption than poly(MPEG350Am) even at low graft densities, presumably due to the increase in PEG content in the grafted layer.

Paclitaxel incorporated in hydrophobically derivatized hyperbranched polyglycerols for intravesical bladder cancer therapy.

OBJECTIVE: To develop paclitaxel incorporated into unimolecular micelles based on hydrophobically derivatized hyperbranched polyglycerols (dHPGs) for use as mucoadhesive intravesical agents against non-muscle-invasive bladder cancer. MATERIALS AND METHODS: Two different types of dHPGs (HPG- C10-polyethylene glycol (PEG) and polyethyleneimine (PEI)-C18-HPG) were synthesized and paclitaxel was loaded into these using a solvent evaporation method. After physicochemical characterization of the resulting nanoparticles, four human bladder cancer cell lines were incubated with various concentrations of paclitaxel incorporated in dHPGs and the results were compared with those of paclitaxel formulated in Cremophor-EL (Taxol(R), Bristol-Myers-Squibb). In vivo, nude mice with orthotopic KU7-luc tumours were intravesically instilled with phosphate buffered saline, Taxol, or paclitaxel/HPG-C10-PEG. RESULTS: dHPGs are mucoadhesive nanoparticles with hydrodynamic radii of <10 nm and incorporation of paclitaxel did not affect their size. The release profiles of paclitaxel from dHPGs were characterized by a rapid-release phase followed by a slower sustained-release phase. While the PEI-C18-HPG formulation released only approximately 40% of the initially incorporated paclitaxel, up to 80% was released from HPG-C10-PEG. Moreover, only paclitaxel/HPG-C10-PEG was stable in acidic urine. In vitro, all paclitaxel formulations potently decreased bladder cancer proliferation although paclitaxel/HPG-C10-PEG was slightly less cytotoxic than standard Taxol. By contrast, in vivo, the mucoadhesive HPG-C10-PEG formulation of paclitaxel was significantly more effective in reducing orthotopic tumour growth than Taxol and was well tolerated. CONCLUSION: Intravesical administration of mucoadhesive nanoparticulate formulations of paclitaxel might be a promising approach for instillation therapy of patients with non-muscle-invasive bladder cancer.

Self-assembled monothiol-terminated hyperbranched polyglycerols on a gold surface: a comparative study on the structure, morphology, and protein adsorption characteristics with linear poly(ethylene glycol)s.

Monothiol-terminated hyperbranched polyglycerols (HPGs) were synthesized by ring-opening polymerization of glycidol from partially deprotonated 2,2'-dihydroxyethane disulfide as the initiator and subsequent reduction of the disulfide group. Two molecular weights of HPG thiols were synthesized. The molecular weights of the polymers were determined by MALDI-TOF analysis, and the presence of thiol was verified by Ellman's assay. The self-assembly of HPG thiols on gold was studied and compared with that of linear poly(ethylene glycol) (PEG) thiols utilizing various surface analysis techniques. Monothiol-functionalized HPGs readily adsorbed to a gold surface and formed highly uniform thin films on the surface. The graft density of the HPG layer decreased with an increase in the molecular weight of the polymer. The amount of polymer on the surface increased with increasing incubation concentration and saturated above 6 g/L polymer concentration. Generally, HPG thiols gave lower graft density compared to linear PEG thiols of similar molecular weight. AFM morphological studies showed that HPG thiols form more uniform and smooth surface films compared to PEG thiols. Incubation of a polymer-coated surface (HPG thiols and PEG thiols) with bovine serum albumin and immunoglobulin showed that the high molecular weight hyperbranched polyglycerol was more resistant to protein adsorption than linear PEG of similar molecular weight or lower molecular weight HPG. The protein adsorption decreased with increasing graft density of the HPG chains on the surface. Our results show that HPG could be a good alternative to PEG in the development of nonfouling functional surfaces.