Stability and nonfouling properties of poly(poly(ethylene glycol) methacrylate) brushes under cell culture conditions

This paper investigates the stability and nonfouling properties of poly(poly(ethylene glycol) methacrylate) (PPEGMA) brushes prepared by surface-initiated atom transfer radical polymerization from SiO(x) substrates modified with a trimethoxysilane-based ATRP initiator. At high chain densities, PPEGMA brushes were found to detach rapidly from glass or silicon substrates. Detachment of the PPEGMA brushes could be monitored with contact angle measurements, which indicated a decrease in the receding water contact angle upon detachment. Detachment of the PPEGMA brushes also resulted in an increase in nonspecific protein adsorption. The stability, and as a consequence the long-term nonfouling properties, of the PPEGMA brushes could be improved by tailoring the brush density and, to a lesser extent, the molecular weight of the polymer chains. By appropriate decrease of the grafting density, the stability of the brushes in cell culture medium could be improved from less than 1 to more than 7 days, without compromising the nonfouling properties.     

Micropatterning of polymer brushes: grafting from dewetting polymer films for biological applications

In this novel platform, a micropatterned polymer brush was obtained by grafting poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMA)) from a thin macroinitiator film using atom transfer radical polymerization (ATRP). A pattern of holes was formed in the macroinitiator film by taking advantage of its spontaneous dewetting above the glass transition temperature from a bottom polystyrene film, driven by unfavorable intermolecular forces. Patterning by dewetting can be achieved at length-scales from a few hundred nanometers to several tens of micrometers, by simply thermally annealing the bilayer above the glass transition temperature of the polymer. This approach is substrate-independent, as polymer films can be cast onto surfaces of different size, shape, or material. As a demonstration of its potential, proteins, and individual cells were attached on targeted bioadhesive polystyrene areas of the micropatterns within poly(PEGMA) protein-repellent brushes. We anticipate this approach will be suitable for the patterning of brushes, especially for biomedical applications such as in the study of single cells and of cell cocultures.     

Shifts in chain-melting transition temperature of liposomal membranes by polymer-grafted lipids

The chain-melting transition temperature of dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was determined by optical turbidity measurements. The dependence on content, Xp, of PEG-DPPE lipid was studied for different polar headgroup sizes, np, of the polymer lipid, throughout the lamellar phase of the mixtures with DPPC. Mean-field theory for the polymer brush regime predicts that the downward shift in transition temperature should vary with polymer size and content as npXp(5/3) (approximately npXp(11/6) for scaling theory). Any shift induced by the charge on PEG-lipids is independent of polymer size. These predictions are reasonably borne out for the longer polymer lipids (PEG molecular masses 750, 2000 and 5000 Da). Transition temperature shifts in the lamellar phase, before the onset of micellisation, are in the region of -1 to -2 degrees C (+/-0.1-0.2 degrees C) in reasonable accord with theoretical estimates of the lateral pressure exerted by the polymer brush. Shifts of this size are significant to the design of liposomes for controlled release of contents by mild hyperthermia.     

Poly(HEMA) brushes emerging as a new platform for direct detection of food pathogen in milk samples

Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 10? cells mL?1 in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 10? cells mL?1, was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG?). However, when the EG? based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples. The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19 nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk.     

Patterned biofunctional poly(acrylic acid) brushes on silicon surfaces

Protein patterning was carried out using a simple procedure based on photolithography wherein the protein was not subjected to UV irradiation and high temperatures or contacted with denaturing solvents or strongly acidic or basic solutions. Self-assembled monolayers of poly(ethylene glycol) (PEG) on silicon surfaces were exposed to oxygen plasma through a patterned photoresist. The etched regions were back-filled with an initiator for surface-initiated atom transfer radical polymerization (ATRP). ATRP of sodium acrylate was readily achieved at room temperature in an aqueous medium. Protonation of the polymer resulted in patterned poly(acrylic acid) (PAA) brushes. A variety of biomolecules containing amino groups could be covalently tethered to the dense carboxyl groups of the brush, under relatively mild conditions. The PEG regions surrounding the PAA brush greatly reduced nonspecific adsorption. Avidin was covalently attached to PAA brushes, and biotin-tagged proteins could be immobilized through avidin-biotin interaction. Such an immobilization method, which is based on specific interactions, is expected to better retain protein functionality than direct covalent binding. Using biotin-tagged bovine serum albumin (BSA) as a model, a simple strategy was developed for immobilization of small biological molecules using BSA as linkages, while BSA can simultaneously block nonspecific interactions.     

Blood clearance and biodistribution of polymer brush-afforded silica particles prepared by surface-initiated living radical polymerization

The physiological properties of polymer brush-afforded silica particles prepared by surface-initiated living radical polymerization were investigated in terms of the circulation lifetime in the blood and distribution in tissues. Hydrophilic polymers consisting mainly of poly(poly(ethylene glycol) methyl ether methacrylate) were grafted onto silica particles by surface-initiated atom transfer radical polymerization that was mediated by a copper complex to produce hairy hybrid particles. A series of hybrid particles was synthesized by varying the diameter of the silica core and the chain length of the polymer brush to examine the relationship between their physicochemical and physiological properties. The hybrid particles were injected intravenously into mice to investigate systematically their blood clearance and body distribution. It was revealed that the structural features of the hybrid particles significantly affected their in vivo pharmacokinetics. Some hybrid particles exhibited an excellently prolonged circulation lifetime in the blood with a half life of ～20 h. When such hybrid particles were injected intravenously into a tumor-bearing mouse, they preferentially accumulated in tumor tissue. The tumor-targeted delivery was optically visualized using hybrid particles grafted with fluorescence-labeled polymer brushes.     

Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol).

The interactive properties of liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are of interest because such liposomes are being developed as drug delivery vehicles and also are ideal model systems for measuring the properties of surface-grafted polymers. For bilayers containing PEG-lipids with PEG molecular weights of 350, 750, 2000, and 5000, pressure-distance relations have been measured by X-ray diffraction analysis of liposomes subjected to known applied osmotic pressures. The distance between apposing bilayers decreased monotonically with increasing applied pressure for each concentration of a given PEG-lipid. Although for bilayers containing PEG-350 and PEG-750 the contribution of electrostatic repulsion to interbilayer interactions was significant, for bilayers containing PEG-2000 and PEG-5000 the major repulsive pressure between bilayers was a steric pressure due to the attached PEG. The range and magnitude of this steric pressure increased both with increasing PEG-lipid concentration and PEG size, and the extension length of the PEG from the bilayer surface at maximum PEG-lipid concentration depended strongly on the size of the PEG, being less than 35 A for PEG-750, and about 65 A for PEG-2000 and 115 A for PEG-5000. The measured pressure-distance relations have been modeled in terms of current theories (deGennes, 1987; Milner et al., 1988b) for the steric pressure produced by surface-grafted polymers, as modified by us to take into account the effects of polymer polydispersity and the possibility that, at low grafting densities, polymers from apposing bilayers surfaces can interpenetrate or interdigitate. No one theoretical scheme is sufficient to account for all the experimental results. However, for a given pressure regime, PEG-lipid size, and PEG-lipid surface density, the appropriately modified theoretical treatment gives a reasonable fit to the pressure-distance data.     

Monomolecular assembly of siRNA and poly(ethylene glycol)-peptide copolymers

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.     

Elastic constants of polymer-grafted lipid membranes

The surface expansion that is induced by the lateral pressure in the brush region of lipid membranes containing grafted polymers is deduced from the scaling and mean-field theories for the polymer brush, together with the equation of state for a lipid monolayer at the equivalence pressure with fluid lipid bilayers. Depending on the length and mole fraction of the polymer lipid, the membrane expansion can be appreciable. Direct experimental evidence for this lateral expansion comes from recent spin-label measurements with lipid membranes containing poly(ethylene glycol)-grafted lipids. The expansion in lipid area modifies the elastic constants of the polymer-grafted membranes in a way that opposes the direct elastic response of the polymer itself. Calculations as a function of polymer lipid content indicate that the net change in isothermal area expansion modulus of the membrane is negative but small, in contrast to previous predictions. A similar situation applies to the curvature elastic moduli of membranes containing short polymer lipids. For longer polymer lipids, however, the direct contribution of the polymer brush to the bending elastic constants dominates, and the increase in bending moduli with increasing polymer lipid content rapidly exceeds the basal values of the bare lipid membrane. The spontaneous (or intrinsic) curvature of the component monolayer of polymer lipid-containing membranes is calculated for the first time. The polymer brush contribution to spontaneous curvature scales quadratically with the polymer length, and at least quadratically with the mole fraction of polymer lipid.     

Substrate-independent approach for the generation of functional protein resistant surfaces

A new route for coating various substrates with antifouling polymer layers was developed. It consisted in deposition of an amino-rich adhesion layer by means of RF magnetron sputtering of Nylon 6,6 followed by the well-controlled, surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by bromoisobutyrate covalently attached to amino groups present in the adhesion layer. Polymer brushes of hydroxy- and methoxy-capped oligoethyleneglycol methacrylate and carboxybetaine acrylamide were grafted from bromoisobutyrate initiator attached to a 15 nm thick amino-rich adhesion layer deposited on gold, silicon, polypropylene, and titanium-aluminum-vanadium alloy surfaces. Well-controlled polymerization kinetics made it possible to control the thickness of the brushes at a nanometer scale. Zero fouling from single protein solutions and a reduction of more than 90% in the fouling from blood plasma observed on the uncoated surfaces was achieved. The feasibility of functionalization with bioactive compounds was tested by covalent attachment of streptavidin onto poly(oligoethylene glycol methacrylate) brush and subsequent immobilization of model antibodies and oligonucleotides. The procedure is nondestructive and does not require any chemical preactivation or the presence of reactive groups on the substrate surface. Contrary to current antifouling modifications, the developed coating can be built on various classes of substrates and preserves its antifouling properties even in undiluted blood plasma. The new technique might be used for fabrication of biotechnological and biomedical devices with tailor-made functions that will not be impaired by fouling from ambient biological media.     

Interaction forces and morphology of a protein-resistant poly(ethylene glycol) layer

The molecular interactions on a protein-resistant surface coated with low-molecular-weight poly(ethylene glycol) (PEG) copolymer brushes are investigated using the extended surface forces apparatus. The observed interaction force is predominantly repulsive and nearly elastic. The chains are extended with respect to the Flory radius, which is in agreement with qualitative predictions of scaling theory. Comparison with theory allows the determination of relevant quantities such as brush length and adsorbed mass. Based on these results, we propose a molecular model for the adsorbed copolymer morphology. Surface-force isotherms measured at high resolution allow distinctive structural forces to be detected, suggesting the existence of a weak equilibrium network between poly(ethylene glycol) and water--a finding in accordance with the remarkable solution properties of PEG. The occurrence of a fine structure is interpreted as a water-induced restriction of the polymer's conformational space. This restriction is highly relevant for the phenomenon of PEG protein resistance. Protein adsorption requires conformational transitions, both in the protein as well as in the PEG layer, which are energetically and kinetically unfavorable.     

Hydraulic conductivity of polymer matrices

We demonstrate that the chondroitin sulfate proteoglycan exhibits enhanced sensitivity to the flow of water compared to other macromolecules which is in accord with their functional role in conferring compressive resistance to cartilage. In order to understand factors that may contribute to its low hydraulic conductivity, a comparative study of hydraulic conductivity, as measured by the sedimentation velocity technique is made of various macromolecules representing variations in charge density, chemical composition, thermodynamic nonideality, size and flexibility. The polymers examined were dextran, poly(ethylene glycol), poly(vinyl alcohol), albumin, and dextran sulfate. The differences in hydraulic conductivity between the various macromolecules could not be explained by conventional theories which included prediction of hydraulic conductivity related to the radius of the molecule regarded as a uniform cylinder, nor the absolute charge density of the molecule and nor to the steric hindrance offered by the macromolecule to the diffusion of tritiated water. A qualitative relationship is established, however, between the noncounterion polymer contribution to osmotic activity and the resistance to water flow for polymers with high osmotic activity.     

Shifts in chain-melting transition temperature of liposomal membranes by polymer-grafted lipids

The chain-melting transition temperature of dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was determined by optical turbidity measurements. The dependence on content, X_p, of PEG-DPPE lipid was studied for different polar headgroup sizes, n_p, of the polymer lipid, throughout the lamellar phase of the mixtures with DPPC. Mean-field theory for the polymer brush regime predicts that the downward shift in transition temperature should vary with polymer size and content as n_pX_p^5/3 (∼n_pX_p^11/6 for scaling theory). Any shift induced by the charge on PEG-lipids is independent of polymer size. These predictions are reasonably borne out for the longer polymer lipids (PEG molecular masses 750, 2000 and 5000 Da). Transition temperature shifts in the lamellar phase, before the onset of micellisation, are in the region of −1 to −2 °C (±0.1-0.2 °C) in reasonable accord with theoretical estimates of the lateral pressure exerted by the polymer brush. Shifts of this size are significant to the design of liposomes for controlled release of contents by mild hyperthermia.     

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.     

Polyelectrolyte brushes grafted from cellulose nanocrystals using Cu-mediated surface-initiated controlled radical polymerization

Herein we report the synthesis of cellulose nanocrystals (CNCs) grafted with poly(acrylic acid) (PAA) chains of different lengths using Cu-mediated surface initiated-controlled radical polymerization (SI-CRP). First, poly(tert-butylacrylate) (PtBA) brushes were synthesized; then, subsequent acid hydrolysis was used to furnish PAA brushes tethered onto the CNC surfaces. The CNCs were chemically modified to create initiator moieties on the CNC surfaces using chemical vapor deposition (CVD) and continued in solvent phase in DMF. A density of initiator groups of 4.6 bromine ester groups/nm(2) on the CNC surface was reached, suggesting a dense functionalization and a promising starting point for the controlled/living radical polymerization. The SI-CRP of tert-butylacrylate proceeded in a well-controlled manner with the aid of added sacrificial initiator, yielding polymer brushes with polydispersity values typically well below 1.12. We calculated the polymer brush grafting density to almost 0.3 chains/nm(2), corresponding to high grafting densities and dense polymer brush formation on the nanocrystals. Successful rapid acid hydrolysis to remove the tert-butyl groups yielded pH-responsive PAA-polyelectrolyte brushes bound to the CNC surface. Individually dispersed rod-like nanoparticles with brushes of PtBA or PAA were clearly visualized by AFM and TEM imaging.     

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.     

A new method for the preparation of giant liposomes in high salt concentrations and growth of protein microcrystals in them

We have developed a new method for the preparation of giant liposomes in aqueous solution containing high salt concentrations (up to 2.0 M). Hydrophilic polymers attached to the surface of lipid membranes by including a small amount of poly(ethylene glycol)-grafted phospholipid in the membrane increase the repulsive force between the membranes, which makes it possible to form giant liposomes at high ionic strength. Using this method, we could grow micron-sized (10-50 μm) protein crystals in a giant liposome. These results demonstrate that this method is a promising tool for the preparation of ‘artificial cells’ under various conditions.     

Interaction of human serum albumin with membranes containing polymer-grafted lipids: spin-label ESR studies in the mushroom and brush regimes

The adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine splitting, 2A(max), of chain spin-labelled phosphatidylcholine in gel-phase membranes. The values of 2A(max) remain constant in the mushroom regime, but decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions X(PEG)(m-->b) approximately 0.04, 0.01-0.02 and 0.005-0.01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of HSA to DPPC membranes is detected as a decrease of the spin label 2A(max) hyperfine splitting in the gel phase. Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the spin-label values of 2A(max) are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively.     

Thermoresponsive micropatterned substrates for single cell studies

We describe the design of micropatterned surfaces for single cell studies, based on thermoresponsive polymer brushes. We show that brushes made of poly(N-isopropylacrylamide) grafted at high surface density display excellent protein and cell anti-adhesive properties. Such brushes are readily patterned at the micron scale via deep UV photolithography. A proper choice of the adhesive pattern shapes, combined with the temperature-dependent swelling properties of PNIPAM, allow us to use the polymer brush as a microactuator which induces cell detachment when the temperature is reduced below [Formula: see text]C.     

Why do Polyethylene Glycol-Coated Liposomes Circulate So Long?: Molecular Mechanism of Liposome Steric Protection with Polyethylene Glycol: Role of Polymer Chain Flexibility

Abstract

The hypothesis is suggested describing the molecular mechanism of protective action of poly(ethylene glycol) on liposomes in vivo on the basis of polymer properties in solvent. The protective layer of polymer on the liposome surface is considered as a “cloud” of possible conformations of macromolecules. If polymer is water-soluble and has flexible main chain, the density of this cloud is high enough to prevent the interaction of opsonins with liposome. At the same time, certain optimal concentration of the protective polymer can be found, when more loose areas in polymeric “clouds” can be used for the immobilization of antibodies on liposomes. As a result, long-circulating targeted liposomes can be obtained.