Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate-co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA-co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(100) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA-co-PEGMEMA) microdomains were activated directly by 1,1'-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.     

Heparin-coupled poly(poly(ethylene glycol) monomethacrylate)-Si(111) hybrids and their blood compatible surfaces

Well-defined (nearly monodispersed) poly(poly(ethylene glycol)monomethacrylate)-Si hybrids were prepared via surface-initiated atom transfer radical polymerization (ATRP) of the poly(ethylene glycol)monomethacrylate (PEGMA) macromonomer on the hydrogen-terminated Si(111) surface (Si-H surface). Both the active chloride groups at the chain ends (from the ATRP process) and the chloride groups converted from some ( approximately 32%) of the -OH groups of the Si-C bonded PEGMA polymer, or P(PEGMA), brushes were used as leaving groups for the covalent coupling of heparin. For the heparinized P(PEGMA)-Si hybrid surfaces, protein adsorption and platelet adhesion were significantly suppressed. The well-defined and dense P(PEGMA) brushes, prepared from surface-initiated ATRP, had allowed the immobilization of a relatively high concentration of heparin (about 14 mug/cm(2)). The resulting silicon surface exhibited significantly improved antithrombogenecity with a plasma recalcification time (PRT) of about 150 min. The persistence of high bioactivity for the immobilized heparin on the hybrid surfaces can be attributed to the biocompatibility of the PEGMA units, as well as their role as spacers in providing the immobilized heparin with a higher degree of conformational freedom in a more hydrophilic environment. Thus, the heparin-coupled P(PEGMA)-Si hybrids with anti-fouling and antithrombogenic surfaces are potentially useful in silicon-based implantable devices and tissue engineering.     

Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery

Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 μg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.     

Functionalized amphiphilic hyperbranched polymers for targeted drug delivery

Amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups were synthesized and characterized. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(epsilon-caprolactone) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by (1)H NMR, (13)C NMR, and combined size-exclusion chromatography and multiangle laser light scattering analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy and particle size analysis. Two antineoplastic drugs, 5-fluorouracil and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drug-loaded nanoparticles to different cells were evaluated in vitro. The results showed the drug-loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug-loaded nanoparticles against folate receptor expressing tumor cells.     

Drug release from and hydrolytic degradation of a poly(ethylene glycol) grafted poly(3-hydroxyoctanoate)

Monoacrylate-poly(ethylene glycol)-grafted poly(3-hydroxyoctanoate) (PEGMA-g-PHO) copolymers were synthesized to develop a swelling-controlled release delivery system for ibuprofen as a model drug. The in vitro hydrolytic degradation of and the drug release from a film made of the PEGMA-g-PHO copolymer were carried out in a phosphate buffer saline (pH 7.4) medium. The hydrolytic degradation of the copolymer was strongly dependent on the degree of grafting (DG) of the PEGMA group. The degradation rate of the copolymer films in vitro increased with increasing DG of the PEGMA group on the PHO chain. The copolymer films showed a controlled delivery of ibuprofen to the medium in periods of time that depend on the composition, hydrophilic/hydrophobic characteristics, initial drug loading amount and film thickness of the graft copolymer support. The drug release rate from the grafted copolymer films was faster than the rate of weight loss of the films themselves. In particular, a combination of the low DG of the PEGMA group in the PHO chains with the low ibuprofen solubility in water led to long-term constant release from these matrices in vitro.     

Depth profile of free volume in a mixture and copolymers of poly(N-vinyl-pyrrolidone) and poly(ethylene glycol) studied by positron annihilation spectroscopy

Effect of hydrogen bonding on the depth profile of the free-volume in a mixture (weight ratio of 65:35) of poly(N-vinyl-pyrrolidone) (PVP) and poly(ethylene glycol) (PEG) and the copolymers of vinyl pyrrolidone with poly(ethylene glycol) diacrylate (PVP-PEGDA) and monomethacrylate (PVP-PEGMMA) was studied using positron annihilation spectroscopy. Doppler broadening energy spectra of annihilation radiation and positron annihilation lifetime were measured as a function of positron incident energy (0-30 keV). Significant variations of the free-volume depth profile in terms of the S parameter, ortho-positronium lifetime, intensity, and lifetime distribution are observed as a result of the hydrogen-bonding replacement of covalent bonds. The polymer mixture with hydrogen bonding through two sides of PEG short chains has a larger free volume and a wider distribution than the comb-structured PVP-PEGMMA and the network structured PVP-PEGDA. A longer ortho-positronium lifetime is observed near the surface than in the bulk. This is interpreted in terms of surface effect, free volume, and hydrogen bonding for drug delivery applications of polymeric materials.     

Influenza hemagglutinin mediated fusion of membranes containing poly(ethylene-glycol) grafted lipids: new insights into the fusion mechanism

The influence of a hydrophilic layer covering the membrane on influenza hemagglutinin (HA) mediated fusion was investigated using membranes containing poly(ethylene-glycol) grafted phosphatidylethanolamine (PEG-2000-PE). Steric inhibition of HA-membrane interactions by these lipids affected virus fusion (half-maximal inhibition at 0.8 mol% for lipids with 114 ethylene glycol residues, or at 3.2 mol% for 45 residues (PEG-2000-PE), concentrations at which the PEG moieties adopt a random coil structure). Reconstituted viral membranes containing 3 mol% PEG-2000-PE retained 40% of their fusion activity. Therefore, efficient fusion is possible with membranes completely covered by a hydrophilic layer of several nanometers, and fusogenic virosomes containing PEG-PE are feasible.     

Polycaprolactone-poly(ethylene glycol) multiblock copolymers as potential substitutes for di(ethylhexyl) phthalate in flexible poly(vinyl chloride) formulations

New high-molecular-weight hydrophobic/hydrophilic segmented copolymers of poly(ester ether carbonate) structure, containing poly(epsilon-caprolactone) (PCL) and poly(ethylene glycol) (PEG) segments in their main chain, were synthesized and characterized. These copolymers were obtained by a two-step chain-extension reaction carried out in the presence of alpha,omega-dihydroxy-oligoPCL of molecular weight 1250 and PEG samples of molecular weight 150, 400, 600, 1000, and 2000. The molecular structures of all synthesized materials were characterized by means of (1)H NMR and (13)C NMR spectroscopy, their molecular weights were determined by means of size exclusion chromatography, and their thermal properties were obtained by means of differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The poly(ester ether carbonate)s of this study are partly or totally miscible at least up to 50 wt % with poly(vinyl chloride) (PVC) and could be used to produce flexible PVC formulations. The miscibility between PVC and the poly(ester ether carbonate)s reported in this paper was investigated by means of DSC and DMA analysis. PVC blends were also analyzed by determining their swellability and the amount of extractables in aqueous media. By comparison purposes, the chain-extension product of PCL1250, that is, PCL polycarbonate, was also synthesized and characterized. The results obtained demonstrated that the copolymers with shortest PEG segment length, i.e. PEG150, 400, and 600, give the best results in terms of miscibility with PVC and lead to blends with maximum resistance to extraction by water. Therefore, they represent, in principle, good substitutes for low-molecular-weight, leachable PVC plasticizers, such as di(ethylhexyl) phthalate.     

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.     

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Reconstruction of surfaces from mixed hydrocarbon and PEG components in water: responsive surfaces aid fouling release

Coatings derived from surface active block copolymers (SABCs) having a combination of hydrophobic aliphatic (linear hydrocarbon or propylene oxide-derived groups) and hydrophilic poly(ethlyene glycol) (PEG) side chains have been developed. The coatings demonstrate superior performance against protein adsorption as well as resistance to biofouling, providing an alternative to coatings containing fluorinated side chains as the hydrophobe, thus reducing the potential environmental impact. The surfaces were examined using dynamic water contact angle, captive air-bubble contact angle, atomic force microscopy, X-ray photoelectron spectroscopy, and near-edge X-ray absorption fine structure analysis. The PS(8K)-b-P(E/B)(25K)-b-PI(10K) triblock copolymer precursor (K3) initially dominated the dry surface. In contrast to previous studies with mixed fluorinated/PEG surfaces, these new materials displayed significant surface changes after exposure to water that allowed fouling resistant behavior. PEG groups buried several nanometers below the surface in the dry state were able to occupy the coating surface after placement in water. The resulting surface exhibits a very low contact angle and good antifouling properties that are very different from those of K3. The surfaces are strongly resistant to protein adsorption using bovine serum albumin as a standard protein challenge. Biofouling assays with sporelings of the green alga Ulva and cells of the diatom Navicula showed the level of adhesion was significantly reduced relative to that of a PDMS standard and that of the triblock copolymer precursor of the SABCs.     

Cationic double-hydrophilic model networks: synthesis,characterization, modeling and protein adsorption studies

Group transfer polymerization (GTP) was used for the preparation of eight networks based on two hydrophilic monomers, 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) methacrylate (PEGMA). Ethylene glycol dimethacrylate (EGDMA) served as the cross-linker, whereas 1,4-bis(methoxytrimethylsiloxymethylene)cyclohexane (MTSMC) was used as a bifunctional initiator. Seven of the networks had linear segments of accurate molecular weight between the cross-links, i.e., they were model networks, whereas the eighth was an equimolar randomly cross-linked network. Five of the seven model networks were based on ABA triblock copolymers with PEGMA midblocks and DMAEMA endblocks, in which the DMAEMA/PEGMA ratio was varied. The remaining two model networks were equimolar isomers, the one based on BAB triblocks (with a DMAEMA midblock) and the other based on the statistical copolymer. The degrees of swelling of all of the networks were measured as a function of pH and were found to increase below pH 7. The degrees of swelling at low pH values increased with the percentage of the DMAEMA monomer, which is ionized under these conditions. These swelling results were confirmed qualitatively by theoretical calculations. Finally, the pH-dependence of the adsorption of the proteins pepsin, bovine serum albumin, and lysozyme onto one of the model networks was studied.     

Biodegradable interpolyelectrolyte complexes based on methoxy poly(ethylene glycol)-b-poly(alpha,L-glutamic acid) and chitosan

We synthesized methoxy poly(ethylene glycol)-b-poly(alpha,L-glutamic acid) (mPEGGA) diblock copolymer by ring-opening polymerization of N-carboxy anhydride of gamma-benzyl-L-glutamate (NCA) using amino-terminated methoxy polyethylene glycol (mPEG) as macroinitiator. Polyelectrolyte complexation between mPEGGA as neutral-block-polyanion and chitosan (CS) as polycation has been scrutinized in aqueous solution as well as in the solid state. Water-soluble polyelectrolyte complexes (PEC) can be formed only under nonstoichiometric condition while phase separation is observed when approaching 1:1 molar mixing ratio in spite of the existence of hydrophilic mPEG block. This is likely due to mismatch in chain length between polyanion block of the copolymer and the polycation or hydrogen bonding between the components. Hydrodynamic size of primary or soluble PEC is determined to be about 200 nm, which is larger than those reported in some literatures. The increase in polyion chain length of the copolymer leads to the increase in the hydrodynamic size of the water-soluble PEC. Formation of spherical micelles by the mPEGGA/CS complex at nonstoichiometirc condition has been confirmed by the scanning electron microscopy observation and transmission electron microscopy observations. The homopolymer CS experiences attractive interaction with both mPEGA and PGA blocks within the copolymer. Competition of hydrogen bonding and electrostatic force in the system or hydrophilic mPEG segments weakens the electrostatic interaction between the oppositely charged polyions. The existence of hydrogen bonding restrains the mobility of mPEG chains of the copolymer and completely prohibits crystallization of mPEG segments. In vitro culture of human fibroblasts indicates that mPEGGA/CS-based materials have potential in biomedical application, especially in tissue engineering.     

Linkage identity is a major factor in determining the effect of PEG-ylated surfactants on permeability of phosphatidylcholine liposomes

The permeability effects induced by single-chained and double-chained poly(ethylene glycol)-surfactants were investigated by measuring the leakage of the fluorescent dye 5(6)-carboxy fluorescein from EPC liposomes. The standard incorporated amount of the surfactants was 5 mol%. Depending on the size of the poly(ethylene glycol) chain and especially on the type of linkage between the polymer and the hydrophobic moiety different leakage profiles were obtained. The presence of a long PEG-polymer resulted in a slower leakage compared with a short analogue. More importantly, the linkage identity was decisive for whether an overall reduction or increase in permeability was obtained. When the hydrocarbon chains were attached to the PEG chain via an ether or an ester the leakage increased compared to pure EPC liposomes. In contrast, if the link was an amide, the leakage was significantly reduced. This effect is assumed to originate from headgroup-headgroup interactions, and most probably hydrogen bonding, between amide and phosphate groups of the PEG-surfactant and the EPC, respectively.     

Preparation and evaluation of polyvinyl alcohol-co-oleylvinyl ether derivatives as tumor-specific cytotoxic systems

A series of poly(vinyl alcohol) amphiphilic derivatives have been prepared to obtain polymeric aggregates in aqueous phase holding thermodynamic instability. The aim was to evaluate their ability to interact with tumor cells eliciting selective cytotoxicity. The poly(vinyl alcohol) derivatives were prepared by partial substitution of poly(vinyl alcohol) (MW 10 kDa) with both oleyl chains and poly(ethylene glycol) monoethyl ethers (PEGMEE) of different molecular weights. The substitution degree was 1.5% for the oleyl chains and 1% for the PEGMEE chains (moles of substituent per 100 mol of hydroxyvinyl monomer). The polyvinyl derivatives obtained easily dissolved in water. Dynamic and static light scattering measurements on the polymer aqueous solutions indicated the formation of polymeric aggregates characterized by low polydispersity (0.232-0.299) and mean size (218-382 nm) in the range suitable for intravenous administration. Moreover, they were characterized by different packing densities and thermodynamic instabilities driving the polymers to interact with hydrophobic membranes. Among the analyzed polymers, the poly(vinyl alcohol)-co-oleylvinyl ether substituted with triethylene glycol monoethyl ether (P10(4)) provided in solution the highest affinity for hydrophobic membranes. P10(4), moreover, was the most cytotoxic toward the tumor cell lines analyzed (neuroblastoma: SH-SY5Y, IMR-32, HTLA-230. melanoma: MZ2-MEL, RPMI7932.), while it did not appreciably alter the viability of the normal resting lymphocytes. The peculiar behavior of the P10(4) aggregates has been correlated to their high thermodynamic instability in solution due to the high packing density that triggers the polymeric aggregates to interact with hydrophobic membranes such as the tumor cell membranes, thus eliciting cytotoxicity.     

An Apparatus for the Concentration of Large Volumes of Dilute Protein Solutions to A Predetermined Volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.     

An apparatus for the concentration of large volumes of dilute protein solutions to a predetermined volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5l) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.     

Contact angle, WAXS, and SAXS analysis of poly(beta-hydroxybutyrate) and poly(ethylene glycol) block copolymers obtained via Azotobacter vinelandii UWD

This study investigated and correlated physical properties and cell interactions of copolymers obtained by a poly(ethylene glycol) (PEG)-modulated fermentation of Azotobacter vinelandii UWD. PEGs with molecular weights of 400 and 3400 Da and di(ethylene glycol) (DEG) were used to modulate the bacterial synthesis of poly(beta-hydroxybutyrate) (PHB). The PHB crystallinity was determined by wide-angle X-ray scattering (WAXS). Small-angle X-ray scattering (SAXS) showed that lamellar distances decreased between the PHB and the PHB modulated with PEG or DEG. Furthermore, the contact angle of water on the PHB/PEG polymer surfaces decreased when compared to that of PHB. The significant decrease of the contact angle and corresponding increase in surface tension, as well as significant decrease in cell adhesion, suggest the presence of hydrophilic PEG and DEG within the hydrophobic surface.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.