Rajesh K. Kainthan

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.

Unimolecular Micelles Based On Hydrophobically Derivatized Hyperbranched Polyglycerols: Ligand Binding Properties

This paper discusses the binding and release properties of hydrophobically modified hyperbranched polyglycerol-polyethylene glycol copolymers that were originally developed as human serum albumin (HSA) substitutes. Their unimolecular micellar nature in aqueous solution has been proven by size measurements and other spectroscopic methods. These polymers aggregate weakly in solution, but the aggregates are broken down by low shear forces or by encapsulating a hydrophobic ligand within the polymer. The small molecule binding properties of these polymers are compared with those of HSA. The preliminary in vitro paclitaxel release studies showed very promising sustained drug release characteristics achieved by these unimolecular micelles.

Hydrophobically derivatized hyperbranched polyglycerol as a human serum albumin substitute

There is a huge clinical demand for Human Serum Albumin (HSA), with a world market of approximately

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

1.5B/year. Concern over prion and viral transmission in the blood supply has led to a need for safer substitutes and offers the opportunity for development of materials with enhanced properties over the presently available plasma expanders. We report here the synthesis and testing of a new synthetic plasma expander that can replace not only the osmotic and volume expansion properties of HSA but, uniquely, its binding and transport properties. We have synthesized several hyperbranched polyglycerols derivatized with hydrophobic groups and short poly(ethylene glycol) (PEG) chains. The hydrophobic groups provide regions for binding fatty acids and other hydrophobic materials while PEG imparts the necessary protection from host defense systems and enhances circulation longevity. These polymers, being hyperbranched, have only a small effect on plasma viscosity. We have shown in vitro that our materials bind 2-3 moles palmitic acid per mole, do not activate the platelet, coagulation or complement systems and do not cause red cell aggregation. In mice these materials are non-toxic with circulation half-lives as high as 34h, controllable by manipulating the molecular weight and the degree of PEG derivatization.

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

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.

The Role of Dimension in Multivalent Binding Events: Structure–Activity Relationship of Dendritic Polyglycerol Sulfate Binding to L-Selectin in Correlation with Size and Surface Charge Density

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.

Branched multifunctional polyether polyketals: variation of ketal group structure enables unprecedented control over polymer degradation in solution and within cells.

L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin-ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC(50) values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure-affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles.

Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols.

Multifunctional biocompatible and biodegradable nanomaterials incorporating specific degradable linkages that respond to various stimuli and with defined degradation profiles are critical to the advancement of targeted nanomedicine. Herein we report, for the first time, a new class of multifunctional dendritic polyether polyketals containing different ketal linkages in their backbone that exhibit unprecedented control over degradation in solution and within the cells. High-molecular-weight and highly compact poly(ketal hydroxyethers) (PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-functionalized AB(2)-type ketal monomers carrying structurally different ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the polymer may play an additional role. This was supported by the log P values of the monomers and the hydrophobicity of the polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation products are highly biocompatible. Taken together, these data demonstrate the utility this new class of biodegradable polymer as a highly promising candidate in the development of multifunctional nanomedicine.

Conjugation to Hyperbranched Polyglycerols Improves RGD-Mediated Inhibition of Platelet Function in Vitro

The covalent attachment of hydrophilic polymers or biopharmaceuticals to the surface of red blood cells (RBCs) has previously been shown as a relatively compatible and effective method for a range of applications. Here, the first example of cell-surface grafting with a hyperbranched and multi-functional macromolecule is described. A range (3 kDa-101 kDa) of dense, globular, and blood compatible hyperbranched polyglycerols (HPG) were synthesized and functionalized with cell-surface reactive, succinimidyl succinate groups (1-12 groups per polymer). Subsequently, HPG was grafted to the RBCs, which were analyzed using physical characterization techniques such as aqueous two-phase partitioning and particle electrophoresis. It was found that the extent of grafting was enhanced by increasing HPG molecular weight, the number of reactive groups per HPG, HPG concentration, and reaction time. Good in vitro cell viability - as measured by lipid peroxidation, hemoglobin oxidation, cell lysis, osmotic fragility, stability in fresh serum and aggregation behavior - was observed for grafting concentrations up to 4.8 mm. The multi-functional aspect of HPG is highlighted by the following observations: using fluorescein-labeled Anti-D (monoclonal) antibody and flow cytometry, the detection of cell-surface Rhesus (RhD) antigens were significantly reduced upon HPG grafting. Secondly, the potential for using HPG as a multi-functional, delivery agent was demonstrated by attaching fluorescent markers to the HPG via degradable linkages prior to grafting.

Long-circulating non-toxic blood pool imaging agent based on hyperbranched polyglycerols

RGD (arginine-glycine-aspartic acid) is a known peptide sequence that binds platelet integrin GPIIbIIIa and disrupts platelet-fibrinogen binding and platelet cross-linking during thrombosis. RGD peptides are unsuitable for clinical applications due to their high 50% inhibitory concentration (IC50) and low in vivo residence times. We addressed these issues by conjugating RGD peptides to biocompatible macromolecular carriers: hyperbranched polyglycerols (HPG) via divinyl sulfone. The GPIIbIIIa binding activity of RGD was maintained after conjugation and the effectiveness of the HPG-RGD conjugate was dependent upon molecular weight and the number of RGD peptides attached to each HPG molecule. These polyvalent inhibitors of platelet aggregation decreased the IC50 of RGD in an inverse linear manner based on the number of RGD peptides per HPG. Since HPG-RGD conjugates do not cause platelet activation by degranulation and certain substitution ratios do not increase fibrinogen binding to resting platelets, HPG-RGD may serve as a model for a novel class of antithrombotics.