Development of soluble ester-linked aldehyde polymers for proteomics.

High molecular weight hyperbranched polyglycerol (HPG) was selected for development as a soluble polymer support for the targeted selection and release of primary-amine containing peptides from a complex mixture. HPG has been functionalized with ester-linked aldehyde groups that can bind primary-amine containing peptides via a reductive alkylation reaction. Once bound, the high molecular weight of the polymer facilitates separation from a complex peptide mixture by employing either a 30 kDa molecular weight cutoff membrane or precipitation in acetonitrile. Following the removal of unbound peptides and reagents, subsequent hydrolysis of the ester linker releases the bound peptide into solution for analysis by mass spectrometry. Released peptides retain the linker moiety and are therefore characteristically mass-shifted. Four water-soluble cleavable aldehyde polymers (CAP1, CAP2, CAP3, and CAP4) ranging in types of linker groups, length of the linker groups, have been prepared and characterized, each demonstrating the ability to selectively enrich and sequence primary-amine peptides from a complex human proteome containing blocked (dimethylated amine) and unblocked (primary amine) peptides. The polymers have very low nonspecific peptide-binding properties while possessing significantly more reactive groups per milligram of the support than commercially available resins. The polymers exhibit a range of reactivities and binding capacities that depend on the type of linker group between the aldehyde group and the polymer. Using various linker structures, we also probed the mechanism of the observed dehydration of hydrolyzed peptides during matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis.

Enhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions.

Macromolecular cell surface modification techniques have shown tremendous utility in various biomedical applications. However, a major drawback concerns inefficient cell surface modification caused by the poor association of hydrophilic macromolecules with cell surfaces. Here, a novel, highly efficient, and universal strategy in which nonreactive "additive" macromolecules are used to modulate the grafting efficiency of cell surface reactive, hydrophilic macromolecules is described. Unprecedented enhanced cell surface modifications by up to 10-fold were observed when various concentrations of a suitable "additive" polymer was present with a constant and low concentration of a "reactive" macromolecule. The importance of this increased efficiency and the possible mechanisms involved are discussed. The cell compatible technique is demonstrated in the case of four different cell types--red blood cells (RBC), leukocytes, platelets, and Jurkat cells. A practical application of grafting macromolecules to cell surfaces in concentrated polymer solutions is demonstrated by the enhanced camouflage of RBC surface antigens for the development of RhD null RBC. In principle, the technique can be adapted to various macromolecular systems and cell types, with significant potential for biomedical applications such as live cell based technologies.

Nonbiofouling polymer brush with latent aldehyde functionality as a template for protein micropatterning.

A novel, nonfouling polymer brush, poly-N-[(2,3-dihydroxypropyl)acrylamide] (PDHPA), containing latent aldehyde groups, was synthesized by surface initiated atom transfer radical polymerization (SI-ATRP). The synthetic parameters were adjusted to produce brushes with varying graft densities and molecular weights. High-density PDHPA brushes successfully prevented the nonspecific protein adsorption from single protein solutions as well as from human platelet poor plasma. Patterns of nonfouling PDHPA and reactive PDHPA-aldehyde domains on the brush surface were created by a combination of photo and wet chemical lithography from a single homogeneous PDHPA brush. Successful micropatterning of single proteins and multiple proteins were achieved using this novel substrate. The high-density brush prevented the diffusion of large proteins into the brush, while a monolayer of covalently coupled proteins was formed on the PDHPA-aldehyde domains. Atomic force microscopy (AFM) force measurements using a biotin coupled AFM tip showed that covalently coupled streptavidin retained its activity, while PDHPA domains showed little nonspecific adsorption of streptavidin. The current study avoids tedious and complicated synthetic processes employed in conventional approaches by providing a novel approach to protein micropatterning from a single, multifunctional polymer brush.

High molecular weight polyglycerol-based multivalent mannose conjugates.

We report the synthesis and characterization of multivalent mannose conjugates based on high molecular weight hyperbranched polyglycerols (HPG). A range of glycoconjugates were synthesized from high molecular weight HPGs (up to 493 kDa) and varying mannose units (22-303 per HPG). Hemagglutination assays using fresh human red blood cells and concanavalin A (Con A) showed that HPG-mannose conjugates exhibited a large enhancement in the relative potency of conjugates (as high as 40000) along with a significant increment in relative activity per sugar (up to 255). The size of the HPG scaffold and the number of mannose residues per HPG were all shown to influence the enhancement of binding interactions with Con A. Isothermal titration calorimetry (ITC) experiments confirmed the enhanced binding affinity and showed that both molecular size and ligand density play important roles. The enhancement in Con A binding to the high molecular weight HPG-mannose conjugates is due to a combination of inter- and intramolecular mannose binding. A few fold increments in the binding constant were obtained over mannose upon covalent attachment to HPG. The binding enhancement is due to the highly favorable entropic contribution to the multiple interactions of Con A to mannose residues on HPG. The high molecular weight HPG-mannose conjugates showed positive cooperativity in binding to Con A. Although carbohydrate density has less of an effect on functional valency of the conjugate compared to the molecular size, it determines the binding affinity.

Enhancement of biological reactions on cell surfaces via macromolecular crowding.

The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors.

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells.

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

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.

In vitro chelating, cytotoxicity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators.

Desferrioxamine (DFO) is used to treat an excess accumulation of iron in the body and is currently the most commonly used iron chelator for the treatment of 'iron overload' disorder. However, the disadvantages of DFO surround its high toxicity and very short plasma half-life. Here, the detailed in vitro evaluation of a novel class of high molecular weight iron chelators based on DFO and polyethylene glycol methacrylate is reported. Reversible addition fragment chain transfer (RAFT) copolymerization afforded polymer conjugates (P-DFO) with well-controlled molecular weight (27-127 kDa) and substitution of DFO (5-26 units per chain) along the copolymer. Human umbilical vein endothelial cell (HUVEC) based cell viability assays showed that the cytotoxicity of P-DFO decreased more than 100-fold at identical concentrations of DFO. The hemocompatibilities of various P-DFO samples were determined by measuring prothrombin time (PT), activated partial thromboplastin time (APTT), thrombelastograph parameters (TEG), complement activation, platelet activation, and red blood cell aggregation. Furthermore, the iron binding properties and chelating efficiency of P-DFO were compared to DFO by measuring the spectral properties upon binding to iron(III), while the prevention of iron(III) mediated oxidation of hemoglobin was also determined. Degradation of the P-DFO conjugates via cleavable ester linkages between the polymer backbone and the PEG side chains was evaluated using gel permeation chromatography (GPC) and NMR. Since the chelating ability of DFO remains intact after conjugation to the copolymer backbone, these macromolecular, blood compatible and degradable conjugates are promising candidates as long circulating, non-toxic iron chelators.