Effect of the polymer chain length of poly(N-isopropylacrylamide) on the temperature-responsive phase transition behavior of its conjugates with [60]fullerene.

In order to develop biomedical materials with specific functionalities, thermoresponsive conjugates [poly(N-isopropylacrylamide)-C(60) (PIPAAm-C(60)) ]of [60]fullerene (C(60)) and PIPAAm with two different polymer chain lengths (4 and 20 kDa) were synthesized by atom transfer radical polymerization. The effects of the polymer chain length on the temperature-responsive phase transition behavior of the synthetic PIPAAm-C(60) conjugates were probed by means of various physicochemical techniques. The coexistence of unimers and molecular assemblies of PIPAAm-C(60) was observed by gel permeation chromatography and dynamic light scattering studies in two PIPAAm-C(60) aqueous solutions below their lower critical solution temperatures (LCSTs). Additionally, below their LCSTs, differences in PIPAAm chain length gave rise to changes in the composition of the unimers and molecular assemblies. In response to temperature, the absorbance of the PIPAAm-C(60) aqueous solution changed according to a two-step behavior profile. Increasing temperature during the primary stage, where a change in the absorbance of the PIPAAm-C(60) aqueous solution took place, did not change the transition temperature, regardless of the solution concentration of PIPAAm-C(60). This absorbance change was associated with the phase transition of the molecular assemblies of PIPAAm-C(60). However, at the second stage, the transition temperature shifted to a higher value with the decrease in the concentration of PIPAAm-C(60), in the same manner as free PIPAAm chains. The second change was associated with the phase transition of the unimeric PIPAAm-C(60). Differences in PIPAAm chain length gave rise to the change in the phase transition behavior of PIPAAm-C(60) aqueous solution. Therefore, the chain length of PIPAAm was found to be a predominant factor involved in the solution characteristics of PIPAAm-C(60). Consequently, the PIPAAm-C(60) is expected to be an intelligent biomaterial possessing heat-induced accumulation and bioactivities.

Attenuation of delayed-type hypersensitivity by fullerene treatment.

Expansion and commercialization of nanotechnology mean that it is important to understand the potential health hazards of manufactured nanoparticles. Here, we focused on the effect of fullerene, a type of nanoparticle already in commercial use, on delayed-type hypersensitivity (DTH) induced by methyl-bovine serum albumin (mBSA). Delayed-type hypersensitivity was induced with methyl-bovine serum albumin in female C57BL/6 mice. A colloidal suspension of crystalline C(60) (nano-C(60); average particle size 165 nm; 200 microL; 5.5 microg/mL) was injected intravenously twice, just before immunization and challenge with mBSA. Nano-C(60) treatment significantly attenuated footpad swelling, compared with that in DTH-disease control mice. Cytokine analysis indicated that nano-C(60) treatment switched the cytokine balance towards Th1-dominance. Pro-inflammatory cytokines IL-6 and IL-17 were significantly increased in DTH mice, and these increases were significantly suppressed by nano-C(60) treatment. Suppression of IL-17 by nano-C(60) was confirmed in an in vitro splenocyte culture. However, production of TNF-alpha was increased in DTH mice, and the increase was significantly enhanced by nano-C(60) treatment. The ratio of regulatory T (Treg) cells to total T (CD4+) cells was also significantly increased by nano-C(60) treatment, compared with that in DTH-disease control mice. Nano-C(60) treatment showed significant immunomodulatory effects in a mouse DTH model: IL-6 and IL-17 production was down-regulated, and the Treg cell ratio was up-regulated, concomitantly with attenuation of the pathology of DTH.

Synthesis of new UV-B light absorbents: (Acetylphenyl)glycosides with antioxidant activities.

m-Acetylphenyl-beta-d-glucopyranosides and m-acetylphenyl-alpha/beta-d-mannopyranosides were synthesized by the Koenigs-Knorr, Mitsunobu, and Helferich reactions as key glycosylation reactions, respectively. Their spectroscopic properties and antioxidative activities were characterized as potential ultraviolet B-ray absorbents.

The use of biotin-avidin binding to facilitate biomodification of thermoresponsive culture surfaces.

Here, we report biomodification of temperature-responsive culture surfaces with biotinylated biomolecules utilizing streptavidin and biotinylation of the surfaces. Poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) was covalently grafted onto tissue culture polystyrene (TCPS) dishes. Biotinylated Arg-Gly-Asp-Ser (RGDS) peptides with different spacer lengths (biotin-conjugated G(n)RGDS (n=1,6,12,16)) were examined. Human umbilical vein endothelial cells (HUVECs) adhered and were well spread on G(12)RGDS-immobilized surfaces in the absence of serum at 37 degrees C, while much less cell adhesion was observed with the other peptides. Adhered HUVECs were detached on reducing temperature to 20 degrees C, or on adding free RGDS peptide. Interestingly, cell detachment was accelerated by applying both these techniques. Consequently, by optimizing the spacer length, biomolecules can be functionally immobilized onto thermoresponsive surfaces via the affinity binding between avidin and biotin.

Creation of a mixed poly(ethylene glycol) tethered-chain surface for preventing the nonspecific adsorption of proteins and peptides.

Using a heterotelechelic poly(ethylene glycol) (PEG) possessing a mercapto group at one end and an acetal group at the other end (acetal-PEG-SH), the authors constructed a reactive PEG tethered-chain surface on a surface plasmon sensor (SPR) gold chip for biosensing with high sensitivity. Nonspecific bovine serum albumin adsorption on the PEG tethered-chain surface was significantly influenced by the density of the PEG chain, and was almost completely suppressed by increasing the PEG density through the repetitive treatment of the chip surface with acetal-PEG-SH. The PEG density was increased even more by adding an underbrushed layer made of shorter-PEG-SH-chain molecules (2 kDa, hereafter 2k) to the surface made of longer-PEG-SH-chain molecules (5 kDa, hereafter 5k). SPR measurement then gave an estimate of the adsorption of a series of proteins with varying sizes and isoelectric points on the PEG chain surface having an underbrushed layer (PEG5k/2k surface). As compared to other SPR surfaces, viz., a commercial carboxymethyl dextran and conventional PEG5k tethered-chain surface without an underbrushed layer, the mixed PEG5k/2k surface showed almost complete inhibition of the nonspecific adsorption not only of high-molecular-weight proteins but also of low-molecular-weight peptides.

A reactive poly(ethylene glycol) layer to achieve specific surface plasmon resonance sensing with a high S/N ratio: the substantial role of a short underbrushed PEG layer in minimizing nonspecific adsorption.

A reactive poly(ethylene glycol) (PEG)-brushed layer was constructed on a surface plasmon resonance (SPR) sensor chip using a heterobifunctional PEG possessing an acetal group at one end and a mercapto group at the other end (alpha-acetal-omega-mercapto-PEG). The density of the PEG brushed layer substantially increased with repetitive adsorption/rinse cycles of the PEG on the sensor chip, allowing dramatic reduction of nonspecific protein adsorption. Notably, formation of a short, filler layer of PEG (2 kDa) in the preconstructed longer PEG brushed layer (5 kDa) achieved almost complete prevention of nonspecific protein adsorption. The acetal group located at the distal end of the tethered PEG was converted to an aldehyde group by the acid treatment, followed by the installation of biocytin hydrazide through Schiff base formation. SPR sensing of streptavidin was done with a very high S/N ratio even in a proteinous medium using the biotinylated PEG (5 kDa) tethered chip with an inert filler layer of short PEG (2 kDa). Furthermore, the specific affinity of streptavidin for the biotinylated PEG was highly influenced by the length of the filler PEG and was significantly reduced when the length of the filler PEG was longer than that of the biotinylated PEG. This result clearly revealed the substantial importance of the steric factor on biospecific interaction at the distal end of tethered PEG on the sensor surface.