JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of
Neuroscience

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of Developmental Biology

You have subscription access to videos in this collection through your user account.

In JoVE (1)

Other Publications (39)

Articles by Donald E. Brooks in JoVE

 JoVE Immunology and Infection

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

1Centre for Blood Research, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia, 3Canadian Blood Services, University of British Columbia, 4Department of Chemistry, Life Sciences Centre, University of British Columbia


JoVE 50075

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Other articles by Donald E. Brooks on PubMed

Evaluation of an Atomic Force Microscopy Pull-off Method for Measuring Molecular Weight and Polydispersity of Polymer Brushes: Effect of Grafting Density

The accuracy of the molecular weights Mn and polydispersities of polymer brushes, determined by stretching the grafted chains using atomic force microscopy (AFM) and measuring the contour length distribution, was evaluated as a function of grafting density sigma. Poly(N,N-dimethylacrylamide) brushes were prepared by surface initiated atom transfer radical polymerization on latex particles with sigma ranging between 0.17 and 0.0059 chains/nm2 and constant Mn. The polymer, which could be cleaved from the grafting surface by hydrolysis and characterized by gel permeation chromatography (GPC), had a Mn of 30,600 and polydispersity (PDI) of 1.35. The Mn determined by the AFM technique for the higher density brushes agreed quite well with the GPC results but was significantly underestimated for the lower sigma. At high grafting density in good solvent, the extended structure of the brush increases the probability of forming segment-tip contacts located at the chain end. When the distance between chains approached twice the radius of gyration of the polymer, the transition from brush to mushroom structure presumably enabled the formation of a larger number of segment-tip contacts having separations smaller than the contour length, which explains the discrepancy between the two methods at low sigma. The PDI was typically higher than that obtained by GPC, suggesting that sampling of chains with above average contour length occurs at a frequency that is greater than their spatial distribution.

The Glycan-rich Outer Layer of the Cell Wall of Mycobacterium Tuberculosis Acts As an Antiphagocytic Capsule Limiting the Association of the Bacterium with Macrophages

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.

Plasma Protein Adsorption to Surfaces Grafted with Dense Homopolymer and Copolymer Brushes Containing Poly(N-isopropylacrylamide)

Growing polymer chains from surface initiators in principle allows much more dense polymer surface layers to be created than can be produced by grafting of whole (self-excluding) chains. We have utilized aqueous atom transfer radical polymerization to graft a series of cleavable hydrophilic poly(N-isopropylacrylamide) (PNIPAM) homopolymers and block copolymers of substituted acrylamides from polystyrene latex to give brushes of controlled MW and surface density. Average chain separations much less than their free solution radii of gyration have been achieved. Exposure to radiolabeled single proteins or to whole plasma and subsequent analysis by SDS-PAGE shows that PNIPAM brushes decrease protein adsorption relative to the latex surface or other substituted polyacrylamides. The PNIPAM brushes exhibit a second-order phase transition around 30 degrees C as reflected by a decrease in the hydrodynamic thickness of the brush at higher temperatures. Total plasma protein adsorption is increased at 40 degrees C compared to 20 degrees C but there is significant differential adsorption behavior among the proteins detected by gel-electrophoresis analysis.

Attractive Bridging Interactions in Dense Polymer Brushes in Good Solvent Measured by Atomic Force Microscopy

Using an atomic force microscope (AFM), we have investigated the interaction forces exerted by latex particles bearing densely grafted polymer brushes consisting of poly(N,N-dimethylacrylamide) (PDMA), poly(methoxyethylacrylamide) (PMEA), poly(N-isopropylacrylamide) (PNIPAM), and PMEA-b-PNIPAM in aqueous media (good solvent). The brushes were prepared by controlled surface-initiated atom transfer radical polymerization, and the hydrodynamic thicknesses were measured by dynamic light scattering. The molecular weight (Mn), grafting density (sigma), and polydispersity (PDI) of the brushes were determined by gel permeation chromatography and multiangle laser light scattering after cleaving the polymer from the latex surface by hydrolysis. Force profiles of PDMA (0.017 nm(-2) < or = sigma < or = 0.17 nm-2) and PMEA (sigma = 0.054 nm-2) brushes were purely repulsive upon compression, with forces increasing with Mn and a, as expected, due to excluded volume interactions. At a sufficiently low grafting density (sigma = 0.012 nm-2), PDMA exhibited a long-range exponentially increasing attractive force followed by repulsion upon further compression. The long-range attractive force is believed to be due to bridging between the free chain ends and the AFM tip. The PNIPAM brush exhibited a bridging force at a grafting density of 0.037 nm(-2), a value lower than the sigma needed to induce bridging in the PDMA brush. Bridging was therefore found to depend on grafting density as well as on the nature of the monomer. The grafting densities of these polymers were larger than those typically associated with bridging. Bridging interactions were used to confirm the presence of PNIPAM in a block copolymer PMEA-b-PNIPAMA brush given that the original PMEA homopolymer brush produced a purely repulsive force. The attractive force was first detected in the block copolymer brush at a separation that increased with the length of the PNIPAM block.

Molecular Weight and Polydispersity Estimation of Adsorbing Polymer Brushes by Atomic Force Microscopy

We have estimated the molecular weight, Mn, and polydispersity, PDI, of densely grafted poly(N-isopropylacrylamide) (PNIPAM) brushes using a novel atomic force microscopy (AFM) approach. When compression of a polymer brush induced adsorption of multiple chains to an AFM tip, the resulting decompression force profile exhibited a maximum attractive force at a separation, Lm, that decayed to zero with increasing tip-sample separation. We have found that the separation Lm approximates the average contour length, Lc, determined by gel permeation chromatography (GPC). The detection of a decaying attractive force at separations larger than Lc suggests that chains of above average length sequentially break free from the tip as they are stretched away from the grafting surface. The shape of the decompression profile in this region approximately paralleled the cumulative weight fraction of the grafted chains determined by GPC. The fraction of chains of a given molecular weight determined from a single force curve fit a log-normal distribution, having a standard deviation that provided an estimate of the PDI. We have characterized two PNIPAM brushes by this AFM technique as well as by GPC coupled to a multiangle laser light-scattering detector (MALLS). The values obtained by AFM-(1) Mn,AFM = (3.8+/-0.5) x 10(4), PDI,(AFM) = 1.3+/-0.1 and (2) Mn,AFM = (9.4+/-1.4) x 10(4), PDI,(AFM) = 1.3+/-0.1-agreed quite well with the corresponding GPC/MALLS values of (1) Mn,GPC = 4.77 x 10(4), PDI,GPC = 1.33 and (2) Mn,GPC = 9.49 x 10(4), PDI = 1.35. This technique requires only a single force curve to obtain a statistical distribution of contour lengths and provides a novel method for estimating the Mn and PDI of appropriate uniformly grafted dense polymer layers.

Entropic Interaction Chromatography: Separating Proteins on the Basis of Size Using End-grafted Polymer Brushes

Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (DeltaSbrush(c)) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that DeltaSbrush(c) is a strong function of both the size (MWp) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of DeltaSbrush(c) on MWp provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a wide-pore (1,000-A pores, 64-microm diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K(P)) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of K(P) values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MWp range is achieved at low to moderate grafting densities and intermediate chain lengths.

Biocompatibility Testing of Branched and Linear Polyglycidol

Polyglycidols are flexible hydrophilic polyethers that are potentially biocompatible polymers based on their similarities to the well-studied poly(ethyleneglycol). Polyglycidols can be prepared as branched or linear polymers by suitable synthetic methods. Biocompatibility testing of these polymers conducted in vitro as well as in vivo are reported here. The in vitro studies included hemocompatibility testing for effects on coagulation (PT and APTT), complement activation, red blood cell aggregation, and whole blood viscosity measurements. In vitro cytotoxicity experiments were also conducted. The results were compared with some of the common biocompatible polymers already in human use. Results from these studies show that polyglycidols are highly biocompatible. Hyperbranched polyglycidols were found to be well tolerated by mice even when injected in high doses.

Blood Compatibility of Novel Water Soluble Hyperbranched Polyglycerol-based Multivalent Cationic Polymers and Their Interaction with DNA

A novel class of hyperbranched polymers based on polyglycerol (PG) and poly(ethylene glycol) (PEG) are synthesized by multibranching anionic ring opening polymerization. Multivalent cationic sites are added to these polymers by a post-amination and quarternization reactions. Blood compatibility studies using these polymers at different concentrations showed insignificant effects on complement activation, platelet activation, coagulation, erythrocyte aggregation and hemolysis compared to branched cationic polyethyleneimine (PEI). The degree of quarternization does not have large influence on the blood compatibility of the new polymers. Cytotoxicity of these polymers is significantly lower than that of PEI and is a function of quarternized nitrogen present in the polymer. Also, these polymers bind DNA in the nanomolar range and are able to condense DNA to highly compact, stable, water soluble nanoparticles in the range of 60-80 nm. Gel electrophoresis studies showed that they form electroneutral complexes with DNA around N/P ratio 1 irrespective of the percentage of quarternization under the conditions studied.

The Influence of Grafted Polymer Architecture and Fluid Hydrodynamics on Protein Separation by Entropic Interaction Chromatography

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 +/- 0.005 chains/nm(2)) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities.

In Vivo Biological Evaluation of High Molecular Weight Hyperbranched Polyglycerols

Hyperbranched polyglycerols (HPGs) are water-soluble polyether polyols that can be synthesized in a controlled manner with low polydispersity. Recently we reported the synthesis and characterization of very high molecular weight and narrowly polydispersed HPGs that could be used as potential alternatives to high generation dendrimers, their advantage being the relative simplicity of synthesis. Reported in this article are the pharmacokinetic properties of these polymers. Two polymers of number average molecular weights 106,000 and 540,000 were tested in mice for their pharmacokinetic behavior. The plasma half-life for the lower molecular weight polymer was around 32 h whereas that of the higher molecular weight HPG was approximately 57 h. Our results show that these high molecular weight HPGs, which can be prepared in a single step reaction, are potential candidates for drug delivery and imaging applications where a long circulating polymer is highly desirable. A detailed tissue distribution profile of these polymers as a function of molecular weight is described. These polymers were also found to be hydrolytically stable and the concentration dependence of solution viscosity measurements suggested the absence of any aggregation.

Synthesis of Novel Size Exclusion Chromatography Support by Surface Initiated Aqueous Atom Transfer Radical Polymerization

We report the use of aqueous surface-initiated atom transfer radical polymerization (SI-ATRP) to grow polymer brushes from a "gigaporous" polymeric chromatography support for use as a novel size exclusion chromatography medium. Poly(N,N-dimethylacrylamide) (PDMA) was grown from hydrolyzable surface initiators via SI-ATRP catalyzed by 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA)/CuCl. Grafted polymer was characterized semiquantitatively by ATR-FTIR and also cleaved and quantitatively characterized for mass, molecular weight, and polydispersity via analytical SEC/MALLS. The synthesis provides control over graft density and allows the creation of dense brushes. Incorporation of negative surface charge was found to be crucial for improving the initiation efficiency. As polymer molecular weight and density could be controlled through reaction conditions, the resulting low-polydispersity grafted polymer brush medium is shown to be suitable for use as a customizable size exclusion chromatography medium for investigating the principals of entropic interaction chromatography. All packed media investigated showed size-dependent partitioning of solutes, even for low graft density systems. Increasing the molecular weight of the grafts allowed solutes more access to the volume fraction in the column available for partitioning. Compared to low graft density media, increased graft density caused eluted solute probes to be retained less within the column and allowed for greater size discrimination of probes whose molecular weights were less than 10(4) kDa.

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 $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.

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.

Unimolecular Micelles Based on Hydrophobically Derivatized Hyperbranched Polyglycerols: Biodistribution Studies

We recently reported the synthesis and testing of a new class of unimolecular micelles based on hyperbranched polyglycerols as second generation synthetic plasma expanders and as general drug delivery vehicles. A detailed biodistribution study of two derivatized hyperbranched polyglycerols of different molecular weights derivatized with hydrophobic groups and short poly(ethylene glycol) chains is reported in this article. In mice, these materials are nontoxic with circulation half-lives as high as 31 h, controllable by manipulating the molecular weight and the degree of PEG derivatization. Organ accumulation is low, presumably due to the "pegylation" effect. Thermal degradation and hydrolysis data suggest that these polymers are highly stable with a long shelf life, a major advantage for a pharmaceutical product. Degradation under acidic conditions has been observed for these polymers.

Paclitaxel Incorporated in Hydrophobically Derivatized Hyperbranched Polyglycerols for Intravesical Bladder Cancer Therapy

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.

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.

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.

Adsorption of Amphiphilic Hyperbranched Polyglycerol Derivatives Onto Human Red Blood Cells

Hydrophobically derivatized hyperbranched polyglycerol (HPG)-polyethylene glycol (PEG) polymers bearing stearoyl chains (HPG-C18-PEG) were originally developed as human serum albumin substitutes and further as a unimolecular drug delivery system. In view of these in vivo applications and the potential for membrane interaction by these materials due to their amphiphilic structure, determining the adsorption of the polymers to human red blood cells (RBCs) is an important issue. This paper reports on the in vitro adsorption to RBCs of tritium-radiolabeled HPG-C18-PEG polymers. The morphological changes of RBCs associated with the adsorption were also examined by light and scanning electron microscopy (SEM). Laser scanning confocal microscopy (LSCM) suggests that the binding site of the polymers on RBCs is the cell membrane. Adsorption experiments show that, in the medium of either saline or plasma, the binding amount of the polymers to RBCs increases with increased polymer concentration in a manner which implies simple Langmurian behavior. The binding amount in saline is of the order of 10(5) molecules/cell at an equilibrium concentration of 1 mg/mL of HPG-C18-PEG polymer. The RBC morphology depends on the adsorbed amount; the cells become crenated in high concentrations (5 and 10 mg/mL) of the polymer solutions in the absence of plasma proteins. Interestingly, a large amount of polymers remain bound to RBCs even after washes with plasma (of the order of 10(4) molecules/cell). Thus, the bound polymers might have an extended circulating time by "hitchhiking" on RBCs in the bloodstream. These results provide significant information and insight for related studies of the interaction of amphiphilic molecules with cell membranes and for in vivo applications of biopolymers as drug delivery systems.

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.

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.

The Induction of Thrombus Generation on Nanostructured Neutral Polymer Brush Surfaces

Surface induced thrombus generation is a major clinical concern associated with vascular medical devices and implants. Here, we show that high graft density hydrophilic non-charged poly (N,N-dimethylacrylamide) (PDMA) brushes prevent the initiation of blood coagulation on synthetic surfaces. Using a multi-faceted analysis approach, we have identified that PDMA brushes greater than 0.27 chains/nm(2) graft density showed this highly desired property. Non-specific protein adsorption is greatly reduced on high density brushes compared to bare surface as evident from isothermal titration calorimetry, gel electrophoresis, and proteomic analyses. We have identified approximately 129 proteins of various types on bare and PDMA brush coated surfaces at a range of surface concentrations. Thromboelastography, platelet activation, and aggregation analyses show that only high graft density brushes are neutral to blood coagulation. Since the polymer brush synthesis can be adapted to most currently used biomedical materials, these results have significant impact in the design of highly hemocompatible surfaces.

The Induction of Thrombus Generation on Nanostructured Neutral Polymer Brush Surfaces

Surface induced thrombus generation is a major clinical concern associated with vascular medical devices and implants. Here, we show that high graft density hydrophilic non-charged poly (N,N-dimethylacrylamide) (PDMA) brushes prevent the initiation of blood coagulation on synthetic surfaces. Using a multi-faceted analysis approach, we have identified that PDMA brushes greater than 0.27 chains/nm2 graft density showed this highly desired property. Non-specific protein adsorption is greatly reduced on high density brushes compared to bare surface as evident from isothermal titration calorimetry, gel electrophoresis, and proteomic analyses. We have identified approximately 129 proteins of various types on bare and PDMA brush coated surfaces at a range of surface concentrations. Thromboelastography, platelet activation, and aggregation analyses show that only high graft density brushes are neutral to blood coagulation. Since the polymer brush synthesis can be adapted to most currently used biomedical materials, these results have significant impact in the design of highly hemocompatible surfaces.

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.

Inhibitory Effect of Hydrophilic Polymer Brushes on Surface-induced Platelet Activation and Adhesion

Poly(N,N-dimethylacrylamide) (PDMA) brushes are successfully grown from unplasticized poly(vinyl chloride) (uPVC) by well-controlled surface-initiated atom transfer radical polymerization (SI-ATRP). Molecular weights of the grafted PDMA brushes vary from ≈ 35,000 to 2,170000 Da, while the graft density ranges from 0.08 to 1.13 chains · nm(-2). The polydispersity of the grafted PDMA brushes is controlled within 1.20 to 1.80. Platelet activation (expression of CD62) and adhesion studies reveal that the graft densities of the PDMA brushes play an important role in controlling interfacial properties. PDMA brushes with graft densities between 0.35 and 0.50 chains · nm(-2) induce a significantly reduced platelet activation compared to unmodified uPVC. Moreover, the surface adhesion of platelets on uPVC is significantly reduced by the densely grafted PDMA brushes. PDMA brushes that have high molecular weights lead to a relatively lower platelet activation compared to low-molecular-weight brushes. However, the graft density of the brush is more important than molecular weight in controlling platelet interactions with PVC. PDMA brushes do not produce any significant platelet consumption in platelet rich plasma. Up to a seven-fold decrease in the number of platelets adhered on high graft density brushes is observed compared to the bare PVC surface. Unlike the bare PVC, platelets do not form pseudopodes or change morphology on PDMA brush-coated surfaces.

Synthesis and Characterization of Carboxylic Acid Conjugated, Hydrophobically Derivatized, Hyperbranched Polyglycerols As Nanoparticulate Drug Carriers for Cisplatin

Hyperbranched polyglycerols (HPGs) with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) were synthesized and further functionalized with carboxylate groups to bind and deliver cisplatin. Low and high levels of carboxylate were conjugated to HPGs (HPG-C(8/10)-MePEG(6.5)-COOH(113) and HPG-C(8/10)-MePEG(6.5)-COOH(348)) and their structures were confirmed through NMR and FTIR spectroscopy and potentiometric titration. The hydrodynamic diameter of the HPGs ranged from 5-10 nm and the addition of COOH groups decreased the zeta potential of the polymers. HPG-C(8/10)-MePEG(6.5)-COOH(113) bound up to 10% w/w cisplatin, whereas HPG-C(8/10)-MePEG(6.5)-COOH(348) bound up to 20% w/w drug with 100% efficiency. Drug was released from HPG-C(8/10)-MePEG(6.5)-COOH(113) over 7 days at the same rate, regardless of the pH. Cisplatin release from HPG-C(8/10)-MePEG(6.5)-COOH(348) was significantly slower than HPG-C(8/10)-MePEG(6.5)-COOH(113) at pH 6 and 7.4, but similar at pH 4.5. Release of cisplatin into artificial urine was considerably faster than into buffer. Carboxylated HPGs demonstrated good biocompatibility, and drug-loaded HPGs effectively inhibited proliferation of KU-7-luc bladder cancer cells.

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.

In Vitro and in Vivo Evaluation of Intravesical Docetaxel Loaded Hydrophobically Derivatized Hyperbranched Polyglycerols in an Orthotopic Model of Bladder Cancer

The objective of this study was to evaluate the tolerability, to establish a dosing regimen, and to evaluate the efficacy of intravesical docetaxel (DTX) formulations in a mouse model of bladder cancer. DTX in commercial formulation (Taxotere, DTX in Tween 80) or loaded in hyperbranched polyglycerols (HPGs) was evaluated. The synthesis and characterization of HPGs with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) in the shell and further functionalized with amine groups (HPG-C(8/10)-MePEG and HPG-C(8/10)-MePEG-NH(2)) is described. Intravesical DTX in either commercial or HPGs formulations (up to 1.0 mg/mL) was instilled in mice with orthotopic bladder cancer xenografts and was well tolerated with no apparent signs of local or systemic toxicities. Furthermore, a single dose of intravesical DTX (0.5 mg/mL) loaded in HPGs was significantly more effective in reducing the tumor growth in an orthotopic model of bladder cancer than the commercial formulation of Taxotere. In addition, DTX-loaded HPG-C(8/10)-MePEG-NH(2) was found to be more effective at lower instillation dose than DTX (0.2 mg/mL)-loaded HPG-C(8/10)-MePEG. Overall, our data show promising antitumor efficacy and safety in a recently validated orthotopic model of bladder cancer. Further research is warranted to evaluate its safety and efficacy in early phase clinical trials in patients refractory to standard intravesical therapy.

In Vivo Evaluation of Mucoadhesive Nanoparticulate Docetaxel for Intravesical Treatment of Non-muscle-invasive Bladder Cancer

The present work describes the development and in vitro and in vivo evaluation of a mucoadhesive nanoparticulate docetaxel (DTX) formulation for intravesical bladder cancer therapy.

The Biocompatibility and Biofilm Resistance of Implant Coatings Based on Hydrophilic Polymer Brushes Conjugated with Antimicrobial Peptides

Bacterial colonization on implant surfaces and subsequent infections are one of the most common reasons for the failure of many indwelling devices. Several approaches including antimicrobial and antibiotic-eluting coatings on implants have been attempted; however, none of these approaches succeed in vivo. Here we report a polymer brush based implant coating that is non-toxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of antimicrobial peptides (AMPs). These tethered AMPs maintained excellent broad spectrum antimicrobial activity in vitro and in vivo. We found that this specially structured robust coating was extremely effective in resisting biofilm formation, and that the biofilm resistance depended on the nature of conjugated peptides. The coating had no toxicity to osteoblast-like cells and showed insignificant platelet activation and adhesion, and complement activation in human blood. Since such coatings can be applied to most currently used implant surfaces, our approach has significant potential for the development of infection-resistant implants.

Bending and Stretching Actuation of Soft Materials Through Surface-initiated Polymerization

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

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.

Antibacterial Surfaces Based on Polymer Brushes: Investigation on the Influence of Brush Properties on Antimicrobial Peptide Immobilization and Antimicrobial Activity

Primary amine containing copolymer, poly(N,N-dimethylacrylamide-co-N-(3-aminopropyl)methacrylamide hydrochloride) (poly(DMA-co-APMA)), brushes were synthesized on Ti surface by surface-initiated atom transfer radical polymerization (SI-ATRP) in aqueous conditions. A series of poly(DMA-co-APMA) copolymer brushes on titanium (Ti) surface with different molecular weights, thicknesses, compositions, and graft densities were synthesized by changing the SI-ATRP reaction conditions. Cysteine-functionalized cationic antimicrobial peptide Tet213 (KRWWKWWRRC) was conjugated to the copolymers brushes using a maleimide-thiol addition reaction after initial modification of the grafted chains using 3-maleimidopropionic acid N-hydroxysuccinimide ester. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM), and ellipsometry analysis. The conjugation of the Tet213 onto brushes strongly depended on graft density of the brushes at different copolymer brush compositions. The peptide density (peptides/nm(2)) on the surface varied with the initial composition of the copolymer brushes. Higher graft density of the brushes generated high peptide density (pepetide/nm(2)) and lower number of peptides/polymer chain and vice versa. The peptide density and graft density of the chains on surface greatly influenced the antimicrobial activity of peptide grafted polymer brushes against Pseudomonas aeruginosa.

Tissue Uptake of Docetaxel Loaded Hydrophobically Derivatized Hyperbranched Polyglycerols and Their Effects on the Morphology of the Bladder Urothelium

Recently, we have reported that docetaxel (DTX) loaded, amine terminated hyperbranched polyglycerol (HPG-C(8/10)-MePEG-NH(2)) nanoparticles significantly increased drug uptake in mouse bladder tissues and was the most effective formulation to significantly inhibit tumor growth in an orthotopic model of bladder cancer. The objective of this study was to investigate the effects of HPG-C(8/10)-MePEG-NH(2) nanoparticles on bladder urothelial morphology and integrity, DTX uptake and permeability in bladder tissue and the extent of bladder urothelial recovery following exposure to, and then washout of, HPG-C(8/10)-MePEG-NH(2) nanoparticles. HPG-C(8/10)-MePEG-NH(2) nanoparticles significantly increased the uptake of DTX in both isolated pig bladder as well as in live mouse bladder tissues. Furthermore, HPG-C(8/10)-MePEG-NH(2) nanoparticles were demonstrated to increase the permeability of the urinary bladder wall by causing changes to the urothelial barrier function and morphology through opening of tight junctions and exfoliation of the superficial umbrella cells. These data suggest that exfoliation may be triggered by an apoptosis mechanism, which was followed by a rapid recovery of the urothelium within 24 h post-instillation of HPG-C(8/10)-MePEG-NH(2) nanoparticles. HPG-C(8/10)-MePEG-NH(2) nanoparticles cause significant but rapidly recoverable changes in the bladder urothelial morphology, which we believe may make them suitable for increasing drug permeability of bladder tissue and intravesical drug delivery.

Long-circulating Non-toxic Blood Pool Imaging Agent Based on Hyperbranched Polyglycerols

Currently, in vivo or in vitro(99m)Tc-radiolabelled red blood cells are the standard blood pool imaging agents. Due to risks associated with handling of blood and the problems with the current (99m)Tc shortage, we were interested in a long-circulating biocompatible synthetic macromolecule that would be simple to prepare and could also be used for PET imaging.

In vivo Circulation, Clearance, and Biodistribution of Polyglycerol Grafted Functional Red Blood Cells

The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs ((3)H-HPG) of different molecular weights; the values ranged from 1 × 10(5) to 2 × 10(6) molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 10(5) HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of (3)H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of (3)H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of (3)H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC.

Hyperbranched Polyglycerols As Trimodal Imaging Agents: Design, Biocompatibility, and Tumor Uptake

Combining various imaging modalities often leads to complementary information and synergistic advantages. A trimodal long-circulating imaging agent tagged with radioactive, magnetic resonance, and fluorescence markers is able to combine the high sensitivity of SPECT with the high resolution of MRI over hours and days. The fluorescence marker helps to confirm the in vivo imaging information at the microscopic level, in the context of the tumor microenvironment. To make a trimodal long-circulating probe, high-molecular-weight hyperbranched polyglycerols (HPG) were modified with a suitable ligand for (111)In radiolabeling and Gd coordination, and additionally tagged with a fluorescent dye. The resulting radiopharmaceutical and contrast agent was nontoxic and hemocompatible. Measured radioactively, its total tumor uptake increased from 2.6% at 24 h to 7.3% at 72 h, which is twice the increase expected due to tumor growth in this time period. Both in vivo MRI and subsequent histological analyses of the same tumors confirmed maximum HPG accumulation at 3 days post injection. Furthermore, Gd-derivatized HPG has an excellent contrast enhancement on T1-weighted MRI at 10× lower molar concentrations than commercially available Galbumin. HPG derivatized with gadolinium, radioactivity, and fluorescence are thus long-circulating macromolecules with great potential for imaging of healthy and leaky blood vessels using overlapping multimodal approaches and for the passive targeting of tumors.

Polyvalent Choline Phosphate As a Universal Biomembrane Adhesive

Phospholipids in the cell membranes of all eukaryotic cells contain phosphatidyl choline (PC) as the headgroup. Here we show that hyperbranched polyglycerols (HPGs) decorated with the 'PC-inverse' choline phosphate (CP) in a polyvalent fashion can electrostatically bind to a variety of cell membranes and to PC-containing liposomes, the binding strength depending on the number density of CP groups per macromolecule. We also show that HPG-CPs can cause cells to adhere with varying affinity to other cells, and that binding can be reversed by subsequent exposure to low molecular weight HPGs carrying small numbers of PCs. Moreover, PC-rich membranes adsorb and rapidly internalize fluorescent HPG-CP but not HPG-PC molecules, which suggests that HPG-CPs could be used as drug-delivery agents. CP-decorated polymers should find broad use, for instance as tissue sealants and in the self-assembly of lipid nanostructures.

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.

Waiting
simple hit counter