Measuring the Interactions between Protein-Coated Microspheres and Polymer Brushes in Aqueous Solutions.

Hydrophilic or zwitterionic polymer-functionalized surfaces have become attractive biomaterials in bioscience and technology due to their excellent protein-resistant ability. Understanding the fundamental interactions between proteins and polymers plays an essential role in the surface design of biomaterials. In this work, we studied the interactions between bovine serum albumin (BSA) and two sorts of polymer brushes including zwitterionic poly(carboxybetaine methacrylate) (PCBMA) and hydrophilic poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) in NaCl aqueous solutions directly with a self-established total internal reflection microscope (TIRM) to provide a better understanding of the underlying nonfouling mechanism of polymers. Our results indicate that both the surface charge and brushes length can affect protein adsorption through electrostatic and steric repulsions, respectively. Both PCBMA- and POEGMA-coated surfaces display negative charge properties due to incomplete coverage and ionic adsorption. As a result, strong electrostatic repulsions between proteins and negatively charged polymer-coated surfaces could contribute to the resistance of protein-coated particles in solutions with low ionic strength (0.1, 0.5, and 1 mM) and disappear in solutions with high ionic strength (10 mM). The measured interaction profiles demonstrate that PCBMA brushes could provide apparent steric forces only at high ionic strength (10 mM), where zwitterionic brushes exhibit a relatively extended conformation with a lack of electrostatic forces between intra- and interpolymers. In contrast, the steric repulsion between proteins and POEGMA brushes appears when particles diffuse at low positions in all salt concentrations (0.1-10 mM) with similar steric decay lengths, which results from the unresponsiveness of POEGMA brushes to the salt stimulus.

Synthesis of iron oxide rods coated with polymer brushes and control of their assembly in thin films.

We investigated the surface-initiated atom transfer radical polymerization (SI-ATRP) of methyl methacrylate (MMA) using monodisperse rod-type particles of iron oxide, ß-FeOOH. The slow hydrolysis of iron(III) chloride yielded monodisperse ß-FeOOH rods with an average length-to-width ratio, L/W, of 6 (L = 210 nm and W = 35 nm on average). The surfaces of the ß-FeOOH rods were modified with a triethoxysilane derivative as an ATRP-initiating site, namely, (2-bromo-2-methyl)propionyloxypropyl triethoxysilane. The SI-ATRP of MMA, mediated by a copper complex, was performed using the initiator-coated ß-FeOOH rods in the presence of a "sacrificial" free initiator. Well-defined poly(methyl methacrylate) (PMMA) brushes with molecular weights of up to 700,000 could be grafted on the ß-FeOOH rods with a surface density as high as 0.3 chains/nm(2). The resultant polymer-brush-afforded hybrid rods exhibited high dispersibility in various solvents for PMMA without forming aggregates. Thin films were prepared by dip-coating from a suspension of the hybrid rods, and the rods were oriented in a specific direction in the films. The arrangement of the rods could be controlled by varying the chain length of the polymer brush and the withdrawal speed during the dip-coating process.

Fabrication of contrast agents for magnetic resonance imaging from polymer-brush-afforded iron oxide magnetic nanoparticles prepared by surface-initiated living radical polymerization.

The aim of this study is to fabricate a contrast agent for magnetic resonance imaging (MRI) by using hybrid particles composed of a core of iron oxide magnetite (Fe3O4) nanoparticles and a shell of hydrophilic polymer brush synthesized by surface-initiated (SI) living radical polymerization. To achieve this, Fe3O4 nanoparticles were surface-modified with initiating groups for atom transfer radical polymerization (ATRP) via a ligand-exchange reaction in the presence of a triethoxysilane derivative having an ATRP initiation site. The ATRP-initiator-functionalized Fe3O4 nanoparticles were used for performing the SI-ATRP of methyl methacrylate to demonstrate the ability of the synthesized nanoparticles to produce well-defined polymer brushes on their surfaces. The polymerization proceeded in a living fashion so as to produce graft polymers with targeted molecular weights and narrow molecular weight distribution. The average graft density was estimated to be as high as 0.7 chains/nm(2), which indicates the formation of so-called concentrated polymer brushes on the Fe3O4 nanoparticles. Dynamic light scattering and transmission electron microscope observations of the hybrid nanoparticles revealed their uniformity and dispersibility in solvents to be excellent. A similar polymerization process was conducted using a hydrophilic monomer, poly(ethylene glycol) methyl ether methacrylate (PEGMA), to prepare Fe3O4 nanoparticles grafted with poly(PEGMA) brushes. The resultant hybrid nanoparticles showed excellent dispersibility in aqueous media including physiological conditions without causing any aggregations. The blood clearance and biodistribution of the hybrid particles were investigated by intravenously injecting particles labeled with a radio isotope, (125)I, into mice. It was found that some hybrid particles exhibited an excellently prolonged circulation lifetime in the blood with a half-life of about 24 h. When such hybrid particles were injected intravenously into a tumor-bearing mouse, they preferentially accumulated in the tumor tissues owing to the so-called enhanced permeability and retention effect. The tumor-targeted delivery was visualized by a T2-enhaced MRI measurement.

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.

A novel approach for UV-patterning with binary polymer brushes.

A mixed self-assembled monolayer (SAM) of an initiator (3-(2-bromo-2-isobutyryloxy)propyl triethoxysilane) for atom transfer radical polymerization (ATRP) and an agent (6-(triethoxysilyl)hexyl 2-(((methylthio)carbonothioyl)thio)-2-phenylacetate) for reversible addition-fragmentation chain transfer (RAFT) polymerization was constructed on the surface of a silicon wafer or glass plate by a silane coupling reaction. When a UV light at 254nm was irradiated at the mixed SAM through a photomask, the surface density of the bromine atom at the end of BPE in the irradiated region was drastically reduced by UV-driven scission of the BrC bond, as observed by X-ray photoelectron spectroscopy. Consequently, the surface-initiated (SI)-ATRP of 2-ethylhexyl methacrylate (EHMA) was used to easily construct the poly(EHMA) (PEHMA) brush domain. Subsequently, SI-RAFT polymerization of a zwitterionic vinyl monomer, carboxymethyl betaine (CMB), was performed. Using the sequential polymerization, the PCMB and PEHMA brush domains on the solid substrate could be very easily patterned. Patterning proteins and cells with the binary polymer brush is expected because the PCMB brush indicated strong suppression of protein adsorption and cell adhesion, and the PEHMA brush had non-polar properties. This technique is very simple and useful for regulating the shape and size of bio-fouling and anti-biofouling domains on solid surfaces.

Polymer-brush-afforded SPIO Nanoparticles Show a Unique Biodistribution and MR Imaging Contrast in Mouse Organs.

INTRODUCTION: To investigate the biodistribution and retention properties of the new super paramagnetic iron oxide (new SPIO: mean hydrodynamic diameter, 100 nm) nanoparticles, which have concentrated polymer brushes in the outer shell and are difficult for phagocytes to absorb, and to compare the new SPIO with clinically approved SPIO (Resovist: mean hydrodynamic diameter, 57 nm). MATERIALS AND METHODS: 16 male C57BL/6N mice were divided in two groups according to the administered SPIO (n = 8 for each group; intravenous injection does, 0.1 ml). In vivo magnetic resonance imaging (MRI) was performed before and one hour, one day, one week and four weeks after SPIO administration by two dimensional-the fast low angle shot (2D-FLASH) sequence at 11.7T. Ex vivo high-resolution images of fixed organs were also obtained by (2D-FLASH). After the ex vivo MRI, organs were sectioned and evaluated histologically to confirm the biodistribution of each particle precisely. RESULTS: The new SPIO was taken up in small amounts by liver Kupffer cells and showed a unique in vivo MRI contrast pattern in the kidneys, where the signal intensity decreased substantially in the boundaries between cortex and outer medulla and between outer and inner medulla. We found many round dark spots in the cortex by ex vivo MRI in both groups. Resovist could be detected almost in the cortex. The shapes of the dark spots were similar to those observed in the new SPIO group. Transmission electron microscopy revealed that Resovist and the new SPIO accumulated in different cells of glomeruli, that is, endothelial and mesangial cells, respectively. CONCLUSION: The new SPIO was taken up in small amounts by liver tissue and showed a unique MRI contrast pattern in the kidney. The SPIO were found in the mesangial cells of renal corpuscles. Our results indicate that the new SPIO may be potentially be used as a new contrast agent for evaluation of kidney function as well as immunune function.

Gradation of proteins and cells attached to the surface of bio-inert zwitterionic polymer brush.

A self-assembled monolayer (SAM) of a 2-bromoisobutyryl end group-carrying initiator for atom transfer radical polymerization (ATRP) was constructed on the surface of silicon wafer or glass substrates via a silane-coupling reaction. When the initiator SAM was irradiated with UV light at 254nm, the surface density of bromine atoms was reduced by the scission of CBr bonds as observed by XPS. With the surface-initiated ATRP of the zwitterionic vinyl monomer, carboxymethyl betaine (CMB), the surface density of PCMB brushes could be easily varied by changing the irradiation period of UV light prior to the polymerization. Furthermore, by using a UV-cut shutter sliding above the initiator SAM-modified substrate at a constant speed, the degree of bromine atom removal could be linearly varied along the direction of movement of the shutter. Consequently, the amount of both proteins adsorbed and cells adhered to the PCMB brush-covered substrate could easily be controlled by the gradation of the surface density of PCMB brushes, which suppressed protein adsorption and cell adhesion. Such a technique is very simple and useful for the regulation of the surface density of adsorbed proteins and adhered cells on an originally bio-inert surface.

Synthesis of silica-polymer core-shell nanoparticles by reversible addition-fragmentation chain transfer polymerization.

Hybrid nanoparticles hold great promise for a range of applications such as drug-delivery vectors or colloidal crystal self-assemblies. The challenge of preparing highly monodisperse particles for these applications has recently been overcome by using living radical polymerization techniques. In particular, the use of reversible addition-fragmentation chain transfer (RAFT), initiated from silica surfaces, yields well-defined particles from a range of precursor monomers resulting in nanoparticles of tailored sizes that are accessible via the rational selection of polymerization conditions. Furthermore, using RAFT allows post-polymerization modification to afford multifunctional, monodisperse, nanostructures under mild and non-stringent reaction conditions.

Sum frequency generation study on the structure of water in the vicinity of an amphoteric polymer brush.

A polyampholyte brush was prepared on the surface of a quartz prism by surface-initiated reversible addition-fragmentation chain-transfer polymerization of methacrylic acid (MA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) at a monomer ratio of 1:1. The sum frequency generation method indicated that water molecules at the amphoteric polymer brush-water interfaces were not greatly oriented in comparison with those at the surfaces of bare quartz prisms, and negatively charged PolyMA and positively charged PolyDMAEMA brushes. The small perturbation effect of an amphoteric polymer brush on the structure of vicinal water was in accordance with the tendency of aqueous solution and thin films of amphoteric polymers observed by using Raman and infrared spectroscopies, respectively.

Structure of water in the vicinity of a zwitterionic polymer brush as examined by sum frequency generation method.

A zwitterionic poly(carboxymethylbetaine) (PCMB) brush was prepared on a fused quartz prism by the surface-initiated atom transfer radical polymerization (SI-ATRP) of CMB monomer. The conformation of PCMB brush and the state of water at the surface of the brush were examined using sum frequency generation (SFG) technique. The C-H stretching band of the brush, indicating the gauche defect of the brush, was affected by the contact medium such as dry nitrogen, water vapor-saturated nitrogen and liquid water. The water molecules at the PCMB-water interfaces were not largely oriented in comparison with the interfacial water of both bare and the ATRP-initiator-modified quartz prisms. The similar tendency was previously observed for water in the vicinity of water-soluble zwitterionic polymers and polymer thin films using Raman and attenuated total reflection (ATR) infrared spectroscopies, respectively. The electrical neutralization between neighboring positive and negative charges might diminish the electrostatic adsorption of water molecules to the vicinity of zwitterionic polymer brushes.

Carboxymethylbetaine copolymer layer covalently fixed to a glass substrate.

A random copolymer of zwitterionic monomer, carboxymethylbetaine (CMB), and 3-methacryloyloxypropyl trimethoxysilane was prepared in ethanol using 2,2'-azobisisobutyronitrile as initiator. The incubation of ethanol solution of the copolymer with a glass plate gave a layer of the copolymer with a thickness of about 2-3 nm. The copolymer-modified glass substrate became highly hydrophilic upon immersion in water, and showed a resistance against non-specific adsorption of proteins, and the degree of resistance increased with the content of CMB residues in the copolymer and leveled off. The adhesion of various cells to the glass substrate was also strongly suppressed by the surface modification with the copolymer layer. Further introduction of PolyCMB graft chains on the surface of the layer enhanced the suppression of cell adhesion due to the steric hindrance for the cells to approach the layer. The usefulness of the combination of zwitterionic polymer layer and graft chains to afford anti-biofouling properties to a solid surface of metal oxides was shown.

Anti-biofouling properties of an amphoteric polymer brush constructed on a glass substrate.

An amphoteric copolymer brush of methacrylic acid (MA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) was prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization using both a free chain transfer agent (n-butylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid) and a radical initiator (4,4'-azobis(4-cyanopentanoic acid)) covalently fixed to a glass substrate. An aqueous solution of the copolymer, Poly(MA-r-DMAEMA), which was simultaneously obtained in liquid phase, had a sufficiently small polydispersity in its molecular weight. The copolymer brush showed effective suppression of non-specific adsorption of bovine serum albumin and egg white lysozyme to the brush. In contrast, both negatively charged PolyMA and positively charged PolyDMAEMA brushes significantly adsorbed the proteins irrespective of their net charges. Upon ion beam irradiation, furthermore, a hollow space with a designed shape could be made on the glass substrate, and both HEK293 and HepG2 cells non-specifically adhered to the space, forming aggregates, while no adhesion to the non-treated area on the brush was observed. These results suggest that the amphoteric polymer brushes will be useful materials for biomedical applications.

Image printing on the surface of anti-biofouling zwitterionic polymer brushes by ion beam irradiation.

A CMB monomer was polymerized on a glass plate with a surface-confined ATRP initiator containing a 2-bromoisobutyryl group. The glass plate modified with a PCMB brush was highly hydrophilic and showed a strong resistance against non-specific adsorption of proteins and cell adhesion. Upon ion beam irradiation, furthermore, the PCMB brush was ablated and a hollow space with a designed shape could be made to which HEK293 cells (from human embryonic kidney) and Hep G2 (from human hepatoma) cells non-specifically adhered, while no adhesion of these cells to the non-treated area on the brush was observed. The present results clearly indicate the usefulness of ion beam-printed patterns of anti-biofouling zwitterionic polymer brushes in the biomedical field.