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Other Publications (39)

Articles by Yuuki Inoue in JoVE

Other articles by Yuuki Inoue on PubMed

Priming of Dolphin Neutrophil Respiratory Burst by Recombinant Tumor Necrosis Factor Alpha

Developmental and Comparative Immunology. Sep, 2002  |  Pubmed ID: 12074931

We studied the effects of recombinant dolphin tumor necrosis factor alpha (rdoTNFalpha) on the respiratory burst activity of dolphin neutrophils. rdoTNFalpha enhanced the luminol-dependent chemiluminescence response of dolphin neutrophils induced by concanavalin-A, opsonized zymosan, and heated plasma, but not that induced by phorbol myristate acetate. The TNF-associated priming activity was concentration- and preincubation time-dependent, and heat-instable. These data suggest that, as in human neutrophils, TNFalpha enhances the respiratory burst in dolphin neutrophils that follows short-term incubation with various receptor-mediated agonists.

Molecular Cloning and Sequencing of the Banded Dogfish (Triakis Scyllia) Interleukin-8 CDNA

Fish & Shellfish Immunology. Mar, 2003  |  Pubmed ID: 12703484

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.

Flow Cytometric Analysis of the Neutrophil Respiratory Burst of Ayu, Plecoglossus Altivelis: Comparison with Other Fresh Water Fish

Fish & Shellfish Immunology. Jul, 2003  |  Pubmed ID: 12787685

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu (Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), eel (Anguilla japonica), and pond smelt (Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3-7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2-19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.

Molecular Cloning and Sequencing of the Silver Chimaera (Chimaera Phantasma) Interleukin-8 CDNA

Fish & Shellfish Immunology. Sep, 2003  |  Pubmed ID: 12892748

Dynamic Motion of Phosphorylcholine Groups at the Surface of Poly(2-methacryloyloxyethyl Phosphorylcholine-random-2,2,2-trifluoroethyl Methacrylate)

Journal of Colloid and Interface Science. Jun, 2004  |  Pubmed ID: 15144818

A series of novel random copolymers composed of hydrophilic and hydrophobic monomer units have been synthesized by a conventional radical polymerization method. As the hydrophilic monomer unit, 2-methacryloyloxyethyl phosphorylcholine (MPC) was selected because the MPC polymers are well known for their excellent bio- and blood compatibilities. The semifluorinated monomer, 2,2,2-trifluoroethyl methacrylate (TFEMA), was used as the hydrophobic monomer. The surface analysis of the copolymer by X-ray photoelectron spectroscopy, dynamic contact angle measurement, and zeta-potential measurement showed that the TFEMA unit was concentrated at the outermost surface in the dry state. The dynamic reorientation of the MPC unit occurred in the wet state because the MPC unit had a strong hydrophilic character. As a result, the monomer unit composition on the surface became almost the same as that in the bulk. Nevertheless, the properties of the surface were hydrophilic in spite of the MPC unit composition of the bulk. In particular, the amount of protein adsorbed on the surface was dramatically reduced when the MPC unit mole fraction was 0.2.

Cell Culture of Clonal Ginbuna Crucian Carp Hematopoietic Cells: Differentiation of Cultured Cells into Erythrocytes in Vivo

Developmental and Comparative Immunology. Jul, 2004  |  Pubmed ID: 15183027

We cultivated kidney hematopoietic cells from clonal triploid ginbuna crucian carp, Carassius auratus langsdorfii. Proliferating cells from hematopoietic cell cultures were harvested and injected into tetraploid hybrids (clonal triploid ginbuna and goldfish hybrid) which possess three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish (Carassius auratus). After injection of cultured triploid cells (donor cells), blood samples were obtained from tetraploid hybrids (recipients) every other week. Blood cells stained with acridine orange were measured by flow cytometry to trace the injected donor cells by means of differences in DNA content. Although erythrocytes were not produced in donor cell cultures, such cultures maintained precursor cells capable of differentiation into erythrocytes in vivo. After 4-12 weeks of transplantation, mature erythrocytes derived from the donors were observed in the blood circulation of the recipient fish. These results indicated that the ginbuna hematopoietic cell culture system is an in vivo situation suitable for the study of hematopoietic control mechanisms.

Molecular Cloning and Sequencing of Japanese Pufferfish (Takifugu Rubripes) NADPH Oxidase CDNAs

Developmental and Comparative Immunology. Jul, 2004  |  Pubmed ID: 15183032

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and cytosolic proteins, p40phox, p47phox and p67phox. In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91phox has three potential N-linked glycosylation sites. gp91phox and p22phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47phox and p67phox have two potential Src homology 3 domains and p40phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91phox, p47phox and p67phox expression were high only in the blood and kidney, while p22phox and p40phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.

Surface Characteristics of Block-type Copolymer Composed of Semi-fluorinated and Phospholipid Segments Synthesized by Living Radical Polymerization

Journal of Biomaterials Science. Polymer Edition. 2004  |  Pubmed ID: 15503632

A series of random and block copolymers composed of hydrophilic and hydrophobic monomer units have been synthesized by the free and living radical polymerization methods, respectively. The hydrophilic monomer unit, 2-methacryloyloxyethyl phosphorylcholine (MPC), was selected because the MPC polymers are well-known for their excellent bio- and blood compatibilities. The semi-fluorinated monomer, 2,2,2-trifluoroethyl methacrylate (TFEMA), was used as the hydrophobic monomer unit. Several analyses of the copolymer surface showed that the TFEMA unit was concentrated at the outermost surface on the random copolymer surface and characteristics of MPC unit was dominant on the block copolymer with a low-MPC-unit composition in the dry state. A reorientation of the MPC unit occurred dynamically in the wet state because of the strong hydrophilicity of the MPC units. In the case of the block copolymer with a low-MPC-unit composition, the surface was covered with the MPC units in the wet state. As a result, the amount of the adsorbed bovine plasma fibrinogen and bovine serum gamma-globulin on the block copolymer surface was reduced dramatically.

Molecular Cloning and Expression Analysis of Rainbow Trout (Oncorhynchus Mykiss) Interleukin-10 CDNAs

Fish & Shellfish Immunology. Apr, 2005  |  Pubmed ID: 15561563

Molecular Cloning and Preliminary Expression Analysis of Banded Dogfish (Triakis Scyllia) CC Chemokine CDNAs by Use of Suppression Subtractive Hybridization

Immunogenetics. Jan, 2005  |  Pubmed ID: 15592825

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP3alpha1 and MIP3alpha2 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 contain four exons and three introns, and MIP3alpha1 and MIP3alpha2 shared the same intron/exon organization with that of human. The MIP3alpha1 and MIP3alpha2 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP3alpha2 gene was much lower than the Trsc-SCYA107 and MIP3alpha1 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP3alpha1 and MIP3alpha2 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP3alpha2 genes in PWBCs was observed at 1-12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP3alpha1 expression was observed at 3-12 h poststimulation only with PMA.

Comparison of Respiratory Burst Activity of Inflammatory Neutrophils in Ayu (Plecoglossus Altivelis) and Carp (Cyprinus Carpio)

Fish & Shellfish Immunology. Oct, 2005  |  Pubmed ID: 15863016

Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

Characteristics of NADPH Oxidase Genes (Nox2, P22, P47, and P67) and Nox4 Gene Expressed in Blood Cells of Juvenile Ciona Intestinalis

Immunogenetics. Aug, 2005  |  Pubmed ID: 16025326

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.

Clathrin-dependent Entry of Severe Acute Respiratory Syndrome Coronavirus into Target Cells Expressing ACE2 with the Cytoplasmic Tail Deleted

Journal of Virology. Aug, 2007  |  Pubmed ID: 17522231

The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.

Molecular Cloning and Preliminary Expression Analysis of Banded Dogfish (Triakis Scyllia) TNF Decoy Receptor 3 (TNFRSF6B)

Fish & Shellfish Immunology. Mar, 2008  |  Pubmed ID: 18201904

Decoy receptor 3 (DcR3), a member of TNF receptor superfamily, is a soluble receptor without death domain and cytoplasmic domain, and secreted by cells and binds with FasL, LIGHT and TL1A. The principal function of DcR3 is the inhibition of apoptosis by the binding cytotoxic ligands. Expression of DcR3 has been reported in a wide array of normal human tissues as well as tumors and tumor cell lines. Recently, DcR3 was reported to modulate a variety of immune responses in mammals. TNFR or DcR3 has been identified in some teleost fishes. However, DcR3 is not reported in cartilaginous fish which is the lowest vertebrate possessing the adaptive immune system. Here we identified DcR3 cDNA in shark (Trsc-DcR3) from an SSH library prepared from peripheral white blood cells stimulated with PMA. Four cysteine-rich domains (CRDs) in common with TNF receptor family members are present in the Trsc-DcR3 sequence. The deduced amino acid sequence of Trsc-DcR3 showed highest identity with the chicken (50.4%), followed by human (46.8%) and rainbow trout (36.5%) DcR3. In a phylogenetic tree of known TNFRSF sequences, the Trsc-DcR3 grouped with the chicken and human DcR3. Trsc-DcR3 mRNA was detected strongly in the gill, moderately in the brain, and weakly in the kidney, thymus and leydig. These data strongly suggest that the gene encoding Trsc-DcR3 in banded dogfish is a homolog of the human gene. mRNA expression of Trsc-DcR3 in the thymus and leydig suggests that DcR3 may act as a modulator in the immune system even at the phylogenetic level of cartilaginous fish.

Gene Transfer Device Utilizing Micron-spiked Electrodes Produced by the Self-organization Phenomenon of Fe-alloy

Lab on a Chip. Jul, 2008  |  Pubmed ID: 18584085

In the diffusional phase transformation of two-phase alloys, the new phase precipitates form the matrix phase at specific temperatures, followed by the formation of a mixed microstructure comprising the precipitate and the matrix. It has been found that by specific chemical-etching treatment, the precipitate in Fe-25Cr-6Ni alloy projects substantially and clusters at the surface. The configuration of the precipitate has an extremely high aspect ratio: it is several microns in width and several tens of microns in length (known as micron-spiked). This study targets the development of a gene transfer device with a micro-spike produced based on the self-organization phenomenon of the Fe-25Cr-6Ni alloy. With this spike-projected device, we tried to efficiently transfer plasmid DNA into adherent cells by electric pulse-triggered gene transfer using a plasmid-loaded electrode (electroporation-based reverse transfection). The spiked structure was applied to a substrate of the device to allow efficient gene transfer into adherent cells, although the general substrate was flat and had a smooth surface. The results suggest that this unique spike-projected device has potential applications in gene transfer devices for the analysis of the human genome in the post-genome period.

Nanoscale Evaluation of Lubricity on Well-defined Polymer Brush Surfaces Using QCM-D and AFM

Colloids and Surfaces. B, Biointerfaces. Nov, 2009  |  Pubmed ID: 19720506

For preparing a "highly lubricated biointerface", which has both excellent lubricity and biocompatibility, we investigated the factors responsible for resistance to friction during polymer grafting. We prepared poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate) (PMMA) brush layers with high graft density and well-controlled thickness using atom transfer radical polymerization (ATRP). We measured the water absorptivity in the polymer brush layers and the viscoelasticity of the polymer-hydrated layers using a quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. The PMPC brush layer had the highest water absorptivity, while the PMPC-hydrated layer had the highest fluidity. The friction properties of the polymer brush layers were determined in air, water, and toluene by atomic force microscopy (AFM). The friction on each polymer brush decreased only when a good solvent was chosen for each polymer. In conclusion, the brush layer possessing high water absorptivity and fluidity in water contributes to reduce friction. PMPC grafting is an effective and promising method for obtaining highly lubricated biointerfaces.

Reduction of Protein Adsorption on Well-characterized Polymer Brush Layers with Varying Chemical Structures

Colloids and Surfaces. B, Biointerfaces. Nov, 2010  |  Pubmed ID: 20705439

To clarify protein adsorption behavior on polymer brush layers, surface characteristics and protein adsorption repellency on polymer brush layers should be precisely determined. Here, we clearly delineated the chemical structure of the polymer brush layers containing various hydrophilic groups, namely, phosphorylcholine, sulfoxybetaine, carboxybetaine (zwitterionic), and hydroxyl group (nonionic) and examined the effects of the chemical structure on initial protein adsorption behavior. Kinetic analysis performed during surface-initiated atom transfer radical polymerization revealed that graft polymerization proceeded in a living manner. The graft density of each type of polymer chain and its surface coverage were high enough to form dense polymer brush structures. The hydroxyl group-bearing polymer brush structure exhibited the highest graft density. Among the zwitterionic polymer brush structures, the graft density and surface coverage of sulfoxybetaine- and carboxybetaine-bearing polymer chains were higher than those of the phosphorylcholine-bearing polymer chains. The amount of protein relative to 100% serum adsorbed on polymer brush layers was quantified using quartz crystal microbalance with dissipation (QCM-D). Protein adsorption on all zwitterionic polymer brush layers apparently decreased with increasing thickness of the grafted polymer layers. Protein adsorption was highly suppressed on thick polymer brush layers bearing phosphorylcholine or sulfoxybetaine groups. However, the amount of proteins adsorbed on thick polymer brush layers bearing hydroxyl groups was 10 times more than that adsorbed on polymer brush layers bearing phosphorylcholine groups. Thus, we concluded that the chemical structure of the polymer brush layer is a significant factor affecting resistance to protein adsorption even for dense polymer brush structures.

Procyanidin B1 Purified from Cinnamomi Cortex Suppresses Hepatitis C Virus Replication

Antiviral Chemistry & Chemotherapy. Aug, 2010  |  Pubmed ID: 20710064

A combination of pegylated interferon and ribavirin is the current standard therapy for hepatitis C virus (HCV) infection, but this combination provides relatively low efficacy, especially in some patients with HCV genotype 1 infection; therefore, the development of novel therapeutic agents is required for further improvement in the treatment of chronic HCV infection.

Novel Polymer Biomaterials and Interfaces Inspired from Cell Membrane Functions

Biochimica Et Biophysica Acta. Mar, 2011  |  Pubmed ID: 20435095

Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials. SCOOP OF REVIEW: One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure.

Preparation and Surface Properties of Polyrotaxane-containing Tri-block Copolymers As a Design for Dynamic Biomaterials Surfaces

Colloids and Surfaces. B, Biointerfaces. Jan, 2012  |  Pubmed ID: 21974908

A tri-block copolymer series containing hydrophilic polyrotaxane and hydrophobic poly(iso-butylmethacrylate) (PiBMA) segments was prepared by atom transfer radical polymerization (ATRP), starting from a pseudopolyrotaxane consisting of 2-bromoisobutyryl end-capped poly(ethylene glycol) (PEG) and α-cyclodextrin (α-CD) and followed by methylation. The dynamic wettability and molecular mobility of the copolymer surfaces were evaluated by dynamic contact angle (DCA) and quartz crystal microbalance with dissipation (QCM-D) measurements, respectively. The polyrotaxane tri-block copolymer surfaces were found to show pronounced dynamic wettability and molecular mobility compared to the control surfaces-a tri-block polymer consisting of PEG and PiBMA, and a PiBMA homopolymer-suggesting that a polyrotaxane loop-like structure exists at the outermost surface in an aqueous environment and exhibits dynamic properties attributable to the possible mobile nature of hydrated α-CD molecules along the PEG backbone. Finally, excellent protein adsorption repellency was achieved on the polyrotaxane tri-block copolymer surface, presumably due to the mobile nature of the supramolecular architecture on the surface.

The Significance of Hydrated Surface Molecular Mobility in the Control of the Morphology of Adhering Fibroblasts

Biomaterials. Apr, 2013  |  Pubmed ID: 23410683

The effects of the hydrated molecular mobility and the surface free energy of polymer surfaces on fibronectin adsorption and fibroblast adhesion were investigated. ABA-type block copolymers composed of polyrotaxane (PRX) with different number of threaded α-cyclodextrin (α-CD), random copolymers with similar chemical composition to the PRX block copolymers, and conventional polymers were prepared to determine a wide range of hydrated molecular mobility (Mf) values estimated by quartz crystal microbalance-dissipation (QCM-D) measurements. Fibronectin adsorption was highly dependent on surface free energy, and high surface fibronectin density resulted in a large projected cell area on the polymer surfaces. However, the morphology of adhering fibroblasts was not explained by the surface free energy, but it was found to be strongly dependent on the Mf values of the polymer surfaces in aqueous media. These results emphasize the importance of Mf in the discussion of the elongated morphology of adhering fibroblasts on various polymer surfaces.

Inducing Rapid Cellular Response on RGD-binding Threaded Macromolecular Surfaces

Journal of the American Chemical Society. Apr, 2013  |  Pubmed ID: 23544620

The rapid response of integrin β1 molecules to an RGD peptide on a dynamic polyrotaxane surface was successfully induced. As a result, RGD peptides introduced on a highly dynamic cyclodextrin molecule enhanced the frequency of contact with specific integrin molecules on the cell membrane at the early stage of material-cell interactions.

Hybridization of Poly(2-methacryloyloxyethyl Phosphorylcholine-block-2-ethylhexyl Methacrylate) with Segmented Polyurethane for Reducing Thrombogenicity

Colloids and Surfaces. B, Biointerfaces. Aug, 2013  |  Pubmed ID: 23563289

Segmented polyurethanes (SPUs) are widely used in biomedical devices owing to their superior mechanical properties. However, their applicability in blood-contacting devices such as small-diameter vascular prostheses is severely limited owing to their thrombogenicity. Thus, it is necessary to develop an SPU material that has a surface with low thrombogenicity. In this study, poly(2-methacryloyloxyethyl phosphorylcholine-block-2-ethylhexyl methacrylate) (B-PMEH) was synthesized as a well-defined diblock copolymer by reversible addition-fragmentation chain transfer (RAFT) polymerization. B-PMEH was hybridized with SPU by the integration of the polymer layers to prepare an SPU/B-PMEH polymer alloy membrane (SB membrane). The MPC units in the B-PMEH phase separated on the immersion of the SB membrane in water, producing a surface that drastically lowered the amount of the absorbed fibrinogen and platelet adhesion as compared to the SPU. Importantly, it was demonstrated that the bulk mechanical properties of the SPU were not affected by the addition of B-PMEH.

A Large Mobility of Hydrophilic Molecules at the Outmost Layer Controls the Protein Adsorption and Adhering Behavior with the Actin Fiber Orientation of Human Umbilical Vein Endothelial Cells (HUVEC)

Journal of Biomaterials Science. Polymer Edition. 2013  |  Pubmed ID: 23796033

Adhesion behaviors of human umbilical vein endothelial cells (HUVECs) are interestingly affected by the mobility of hydrophilic chains on the material surfaces. Surfaces with different molecular mobilities were prepared using ABA-type block copolymers consisting polyrotaxane (PRX) or poly(ethylene glycol) (PEG) central block (A block), and amphiphilic anchoring B blocks of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). Two different molecular mobilities of the PRX chains were designed by using normal α-cyclodextrin (α-CD) or α-CD whose hydroxyl groups were converted to methoxy groups in a given ratio to improve its molecular mobility (PRX-PMB and OMe-PRX-PMB). The surface mobility of these materials was assessed as the mobility factor (Mf), which is measured by quartz crystal microbalance with dissipation monitoring system. HUVECs adhered on OMe-PRX-PMB surface much more than PRX-PMB and PMB-block-PEG-block-PMB (PEG-PMB) surfaces. These different HUVEC adhesions were correlated with the density of cell-binding site of adsorbed fibronectin. In addition, the alignment of the actin cytoskeleton of adhered HUVECs was strongly suppressed on the PEG-PMB, PRX-PMB, and OMe-PRX-PMB in response to the increased Mf value. Remarkably, the HUVECs adhered on the OMe-PRX-PMB surface with much less actin organization. We concluded that not only the cell adhesion but also the cellular function are regulated by the molecular mobility of the outmost material surfaces.

Elastic Repulsion from Polymer Brush Layers Exhibiting High Protein Repellency

Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2013  |  Pubmed ID: 23898820

Hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(2-hydroxyethyl methacrylate) (PHEMA) brush layers with different thicknesses and graft densities were prepared to construct a model surface to elucidate protein-surface interactions. In particular, we focused on the steric repulsion of hydrophilic polymer layers as one of the surface properties that strongly influence protein adsorption and employed force-versus-distance (f-d) curve measurements obtained via atomic force microscopy to quantitatively evaluate the steric repulsion force, which is also referred to as the "elastic repulsion energy." We also analyzed direct interactions between the surface and proteins via the f-d curve, because these interactions trigger the protein-adsorption phenomenon. Protein-surface interactions were extremely suppressed at surfaces with high elastic repulsion energies and highly dense polymer brush structures, which is in contrast to those at surfaces with low elastic repulsion energies and low density of the grafted polymer layers. These results indicate that the elastic repulsion from the grafted polymer layer at the surface is an important parameter for controlling protein-surface interactions and protein adsorption phenomenon.

Effects of Molecular Architecture of Phospholipid Polymers on Surface Modification of Segmented Polyurethanes

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 24417469

To modify the surface properties of segmented polyurethane (SPU), effects of the molecular architecture of the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers on the performance of the SPU/MPC polymer membrane were investigated. We combined the random-type, block-type, and graft-type of the MPC polymers with a typical SPU, Tecoflex(®) using double solution casting procedure. The graft-type MPC polymers composed of a poly(MPC) main chain and poly(2-ethylhexyl methacrylate (EHMA)) side chains were synthesized through the combination of two different living radical polymerization techniques to regulate the density and chain length of the side chains. The SPU membranes modified with the MPC polymers were characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The results revealed that the MPC units were located on the SPU surface. Although the breaking strength of the SPU membranes modified with block-type poly(MPC-block-EHMA) and graft-type poly(MPC-graft-EHMA) was lower than that of SPU membranes modified with random-type poly(MPC-random-EHMA), their breaking strengths were adequate for manufacturing medical devices. On the other hand, better stability was observed in the MPC polymer layer on the SPU membrane after immersion in an aqueous medium, wherein the SPU membrane had been modified with the poly(MPC-graft-EHMA). This was because of the intermixing of the hydrophobic poly(EHMA) segments in the domain of the hard segments in the SPU membrane. After this modification, each SPU/MPC polymer membrane showed hydrophilic nature based on the MPC polymers and a dramatic suppression of protein adsorption. From these results, we concluded that the SPU membrane modified with the poly(MPC-graft-EHMA) was one of the promising polymeric biomaterials for making blood-contacting medical devices.

Evaluation of the Durability and Antiadhesive Action of 2-methacryloyloxyethyl Phosphorylcholine Grafting on an Acrylic Resin Denture Base Material

The Journal of Prosthetic Dentistry. Aug, 2014  |  Pubmed ID: 24461942

The polymer 2-methacryloyloxyethyl phosphorylcholine is currently used on medical devices to prevent infection. Denture plaque-associated infection is regarded as a source of serious dental and medical complications in the elderly population, and denture hygiene, therefore, is an issue of considerable importance for denture wearers. Furthermore, because denture bases are exposed to mechanical stresses, for example, denture brushing, the durability of the coating is important for retaining the antiadhesive function of 2-methacryloyloxyethyl phosphorylcholine.

Quantitative Evaluation of Interaction Force Between Functional Groups in Protein and Polymer Brush Surfaces

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2014  |  Pubmed ID: 24564418

To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be a suitable parameter for understanding protein adsorption.

Durable Modification of Segmented Polyurethane for Elastic Blood-contacting Devices by Graft-type 2-methacryloyloxyethyl Phosphorylcholine Copolymer

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 24894706

We propose a novel application of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers for enhancing the performance of modified segmented polyurethane (SPU) surfaces for the development of a small-diameter vascular prosthesis. The SPU membranes were modified by random-type, block-type, and graft-type MPC polymers that were prepared using a double-solution casting procedure on stainless steel substrates. Among these MPC polymers, the graft-type poly(MPC-graft-2-ethylhexyl methacrylate [EHMA]), which is composed of a poly(MPC) segment as the main chain and poly(EHMA) segments as side chains, indicated a higher stability on the SPU membrane after being peeled off from the stainless steel substrate, as well as after immersion in an aqueous medium. This stability was caused by the intermiscibility in the domain of the poly(EHMA) segments and the soft segments of the SPU membrane. Each SPU/MPC polymer membrane exhibited a dramatic suppression of protein adsorption from human plasma and endothelium cell adhesion. Based on these results, the performance of SPU/poly(MPC-graft-EHMA) tubings 2 mm in diameter as vascular prostheses was investigated. Even after blood was passed through the tubings for 2 min, the graft-type MPC polymers effectively protected the blood-contacting surfaces from thrombus formation. In summary, SPU modified by graft-type MPC polymers has the potential for practical application in the form of a non-endothelium, small-diameter vascular prosthesis.

Quantitative Evaluation of Interaction Force of Fibrinogen at Well-defined Surfaces with Various Structures

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25025547

The effects of functional groups and structures at the surface of biomaterials on protein adsorption were examined using direct interaction force measurements. Three kinds of surface structures were evaluated: polymer brushes, self-assembled monolayers with low molecular weight compounds, and surfaces with conventional polymer coatings. These surfaces had various functional groups including phosphorylcholine (PC) group. The surface characterization demonstrated that surface wettability and flexibility depended on both the structure of the surface and the functional groups at the surface. The interactions of protein with these surfaces were evaluated by a force vs. distance curve using an atomic force microscope (AFM). We used fibrinogen as the protein, and the fibrinogen was immobilized on the surface of the AFM cantilever by a conventional technique. It was observed that the interaction force of fibrinogen was strongly related to surface hydrophobic nature and flexibility. That is, the interaction force increased with the increasing hydrophobic nature of the surface. The relationship between the amount of fibrinogen adsorbed on the surface and the interaction force showed good correlation in the range of fibrinogen adsorption from 0 to 250 ng/cm(2), that is, in a monolayered adsorption region. The interaction force decreased with increasing surface viscoelasticity. The most effective surface for preventing fibrinogen adsorption was the polymer brush surface with phosphorylcholine (PC) groups, that is, poly(2-methacryloyloxyethyl phosphorylcholine) brush. The interaction force of this sample was less than 0.1 nN and the amount of fibrinogen adsorbed on the surface was minimal. It was found that the evaluation of protein adsorption based on the interaction force measurement is useful for low-protein adsorption surfaces. It was demonstrated that an extremely hydrophilic and flexible surface could weaken the protein interactions at the surface, resulting in greater resistance to protein adsorption.

Mobility of the Arg-Gly-Asp Ligand on the Outermost Surface of Biomaterials Suppresses Integrin-mediated Mechanotransduction and Subsequent Cell Functions

Acta Biomaterialia. Feb, 2015  |  Pubmed ID: 25463493

Mechanotransduction in the regulation of cellular responses has been previously studied using elastic hydrogels. Because cells interact only with the surface of biomaterials, we are focusing on the molecular mobility at the outermost surface of biomaterials. In this study, surfaces with the mobile Arg-Gly-Asp-Ser (RGDS) peptide have been constructed. Cell culture substrates were coated with ABA-type block copolymers composed of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) segments (A) and a polyrotaxane (PRX) unit with RGDS bound to α-cyclodextrin (B). Adhesion, morphological changes and actin filament formation of human umbilical vein endothelial cells were reduced on the surfaces containing mobile PRX-RGDS in comparison to the immobile RGDS surfaces constructed from random copolymers with RGDS side groups (Prop-andom-RGDS). In the neurite outgrowth assay using rat adrenal pheochromocytoma cells (PC12), only ∼20% of adherent PC12 cells had neurites on PRX-RGDS surfaces, but more than 50% did on the Random-RGDS surface. The beating colony of dimethyl-sulfoxide-treated mouse embryonic carcinoma cells (P19CL6) were found 10 and 14 days after induction on PRX-RGDS and Random-RGDS surfaces, respectively. After 22 days, the beating colony disappeared on PRX-RGDS surfaces, but many colonies remained on Random-RGDS surfaces. These data suggest that the molecular mobility of the cell-binding ligand on the outermost surface of materials effectively suppresses the actin filament formation and differentiation of these functional cell lines, and may be used as a culture substrate for immature stem cells or progenitor cells.

Molecular Interaction Forces Generated During Protein Adsorption to Well-defined Polymer Brush Surfaces

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2015  |  Pubmed ID: 25719761

The molecular interaction forces generated during the adsorption of proteins to surfaces were examined by the force-versus-distance (f-d) curve measurements of atomic force microscopy using probes modified with appropriate molecules. Various substrates with polymer brush layers bearing zwitterionic, cationic, anionic, and hydrophobic groups were systematically prepared by surface-initiated atom transfer radical polymerization. Surface interaction forces on these substrates were analyzed by the f-d curve measurements using probes with the same polymer brush layer as the substrate. Repulsive forces, which decreased depending on the ionic strength, were generated between cationic or anionic polyelectrolyte brush layers; these were considered to be electrostatic interaction forces. A strong adhesive force was detected between hydrophobic polymer brush layers during retraction; this corresponded to the hydrophobic interaction between two hydrophobic polymer layers. In contrast, no significant interaction forces were detected between zwitterionic polymer brush layers. Direct interaction forces between proteins and polymer brush layers were then quantitatively evaluated by the f-d curve measurements using protein-immobilized probes consisting of negatively charged albumin and positively charged lysozyme under physiological conditions. In addition, the amount of protein adsorbed on the polymer brush layer was quantified by surface plasmon resonance measurements. Relatively large amounts of protein adsorbed to the polyelectrolyte brush layers with opposite charges. It was considered that the detachment of the protein after contact with the polymer brush layer hardly occurred due to salt formation at the interface. Both proteins adsorbed significantly on the hydrophobic polymer brush layer, which was due to hydrophobic interactions at the interface. In contrast, the zwitterionic polymer brush layer exhibited no significant interaction force with proteins and suppressed protein adsorption. Taken together, our results suggest that to obtain the protein-repellent surfaces, the surface should not induce direct interaction forces with proteins after contact with them.

Preparation of Photoreactive Phospholipid Polymer Nanoparticles to Immobilize and Release Protein by Photoirradiation

Colloids and Surfaces. B, Biointerfaces. Nov, 2015  |  Pubmed ID: 26263222

Photoreactive and cytocompatible polymer nanoparticles for immobilizing and releasing proteins were prepared. A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-4-(4-(1-methacryloyloxyethyl)-2-methoxy-5-nitrophenoxy) butyric acid (PL)) (PMB-PL) was synthesized. The PMB-PL underwent a cleavage reaction at the PL unit with photoirradiation at a wavelength of 365 nm. Additionally, the PMB-PL took polymer aggregate in aqueous medium and was used to modify the surface of biodegradable poly(L-lactic acid) (PLA) nanoparticle as an emulsifier. The morphology of the PMB-PL/PLA nanoparticle was spherical and approximately 130 nm in diameter. The carboxylic acid group in the PL unit could immobilize proteins by covalent bonding. The bound proteins were released by a photoinduced cleavage reaction. Within 60s, up to 90% of the immobilized proteins was released by photoirradiation. From these results and with an understanding of the fundamental properties of MPC polymers, we concluded that PMB-PL/PLA nanoparticles have the potential to be used as smart carriers to deliver proteins to biological systems, such as the inside of living cells.

Surface Functionalization of Quantum Dots with Fine-structured PH-sensitive Phospholipid Polymer Chains

Colloids and Surfaces. B, Biointerfaces. Nov, 2015  |  Pubmed ID: 26283498

To add novel functionality to quantum dots (QDs), we synthesized water-soluble and pH-responsive block-type polymers by reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers were composed of cytocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer segments, which contain a small fraction of active ester groups and can be used to conjugate biologically active compounds to the polymer, and pH-responsive poly(2-(N,N-diethylamino) ethyl methacrylate (DEAEMA)) segments. One terminal of the polymer chain had a hydrophobic alkyl group that originated from the RAFT initiator. This hydrophobic group can bind to the hydrophobic layer on the QD surface. A fluorescent dye was conjugated to the polymer chains via the active ester group. The block-type polymers have an amphiphilic nature in aqueous medium. The polymers were thus easily bound to the QD surface upon evaporation of the solvent from a solution containing the block-type polymer and QDs, yielding QD/fluorescence dye-conjugated polymer hybrid nanoparticles. Fluorescence resonance energy transfer (FRET) between the QDs (donors) and the fluorescent dye molecules (acceptors) was used to obtain information on the conformational dynamics of the immobilized polymers. Higher FRET efficiency of the QD/fluorescent dye-conjugated polymer hybrid nanoparticles was observed at pH 7.4 as compared to pH 5.0 due to a stretching-shrinking conformational motion of the poly(DEAEMA) segments in response to changes in pH. We concluded that the block-type MPC polymer-modified nanoparticles could be used to evaluate the pH of cells via FRET fluorescence based on the cytocompatibility of the MPC polymer.

Preparation of a Thick Polymer Brush Layer Composed of Poly(2-methacryloyloxyethyl Phosphorylcholine) by Surface-initiated Atom Transfer Radical Polymerization and Analysis of Protein Adsorption Resistance

Colloids and Surfaces. B, Biointerfaces. May, 2016  |  Pubmed ID: 26896657

The purpose of this study was to prepare a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) and assess its resistance to protein adsorption from the dissolved state of poly(MPC) chains in an aqueous condition. The thick poly(MPC) brush layer was prepared through the surface-initiated atom transfer radical polymerization (SI-ATRP) of MPC with a free initiator from an initiator-immobilized substrate at given [Monomer]/[Free initiator] ratios. The ellipsometric thickness of the poly(MPC) brush layers could be controlled by the polymerization degree of the poly(MPC) chains. The thickness of the poly(MPC) brush layer in an aqueous medium was larger than that in air, and this tendency became clearer when the polymerization degree of the poly(MPC) increased. The maximum thickness of the poly(MPC) brush layer in an aqueous medium was around 110 nm. The static air contact angle of the poly(MPC) brush layer in water indicated a reasonably hydrophilic nature, which was independent of the thickness of the poly(MPC) brush layer at the surface. This result occurred because the hydrated state of the poly(MPC) chains is not influenced by the environment surrounding them. Finally, as measured with a quartz crystal microbalance, the amount of protein adsorbed from a fetal bovine serum solution (10% in phosphate-buffered saline) on the original substrate was 420 ng/cm(2). However, the poly(MPC) brush layer reduced this value dramatically to less than 50 ng/cm(2). This effect was independent of the thickness of the poly(MPC) brush layer for thicknesses between 20 nm and about 110 nm. These results indicated that the surface covered with a poly(MPC) brush layer is a promising platform to avoid biofouling and could also be applied to analyze the reactions of biological molecules with a high signal/noise ratio.

High-efficiency Preparation of Poly(2-methacryloyloxyethyl Phosphorylcholine) Grafting Layer on Poly(ether Ether Ketone) by Photoinduced and Self-initiated Graft Polymerization in an Aqueous Solution in the Presence of Inorganic Salt Additives

Acta Biomaterialia. Aug, 2016  |  Pubmed ID: 27154499

A highly efficient methodology for preparing a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) layer on the surface of poly(ether ether ketone) (PEEK) was examined by photoinduced and self-initiated graft polymerization. To enhance the polymerization rate, we demonstrated the effects of inorganic salt additives in the feed monomer solution on thickness of grafted PMPC layer. Photoinduced polymerization occurred and the PMPC graft layer was successfully formed on the PEEK surface, regardless of inorganic salt additives. Moreover, it was clearly observed that the addition of inorganic salt enhanced the grafting thickness of PMPC layer on the surface even when the photoirradiation time was shortened. The addition of inorganic salt additives in the feed monomer solution enhanced the polymerization rate of MPC and resulted in thicker PMPC layers. In particular, we evaluated the effect of NaCl concentration and how this affected the polymerization rate and layer thickness. We considered that this phenomenon was due to the hydration of ions in the feed monomer solution and subsequent apparent increase in the MPC concentration. A PMPC layer with over 100-nm-thick, which was prepared by 5-min photoirradiation in 2.5mol/L inorganic salt aqueous solution, showed good wettability and protein adsorption resistance compared to that of untreated PEEK. Hence, we concluded that the addition of NaCl into the MPC feed solution would be a convenient and efficient method for preparing a graft layer on PEEK.

Precise Control of Surface Electrostatic Forces on Polymer Brush Layers with Opposite Charges for Resistance to Protein Adsorption

Biomaterials. Oct, 2016  |  Pubmed ID: 27512944

Various molecular interaction forces are generated during protein adsorption process on material surfaces. Thus, it is necessary to control them to suppress protein adsorption and the subsequent cell and tissue responses. A series of binary copolymer brush layers were prepared via surface-initiated atom transfer radical polymerization, by mixing the cationic monomer unit and anionic monomer unit randomly in various ratios. Surface characterization revealed that the constructed copolymer brush layers exhibited an uniform super-hydrophilic nature and different surface potentials. The strength of the electrostatic interaction forces operating on these mixed-charge copolymer brush surfaces was evaluated quantitatively using force-versus-distance (f-d) curve measurements by atomic force microscopy (AFM) and probes modified by negatively charged carboxyl groups or positively charged amino groups. The electrostatic interaction forces were determined based on the charge ratios of the copolymer brush layers. Notably, the surface containing equivalent cationic/anionic monomer units hardly interacted with both the charged groups. Furthermore, the protein adsorption force and the protein adsorption mass on these surfaces were examined by AFM f-d curve measurement and surface plasmon resonance measurement, respectively. To clarify the influence of the electrostatic interaction on the protein adsorption behavior on the surface, three kinds of proteins having negative, positive, and relatively neutral net charges under physiological conditions were used in this study. We quantitatively demonstrated that the amount of adsorbed proteins on the surfaces would have a strong correlation with the strength of surface-protein interaction forces, and that the strength of surface-protein interaction forces would be determined from the combination between the properties of the electrostatic interaction forces on the surfaces and the charge properties of the proteins. Especially, the copolymer brush surface composed of equivalent cationic/anionic monomer units exhibited no significant interaction forces, and dramatically suppressed the adsorption of proteins regardless of their charge properties. We conclude that the established methodology could elucidate relationship between the protein adsorption behavior and molecular interaction, especially the electrostatic interaction forces, and demonstrated that the suppression of the electrostatic interactions with the ionic functional groups would be important for the development of new polymeric biomaterials with a high repellency of protein adsorption.

Movement of a Quantum Dot Covered with Cytocompatible and PH-Responsible Phospholipid Polymer Chains Under a Cellular Environment

Biomacromolecules. Dec, 2016  |  Pubmed ID: 27791358

Quantum dots (QDs) were functionalized with well-defined polymer chains having both cytocompatibility and pH-responsiveness to monitor the movement of nanoparticles in a cellular environment with changing local pH. We used a triblock-type water-soluble polymer composed of three segments: (1) a pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate; DEAEMA] segment, (2) a poly[ω-(p-nitrophenyloxycarbonyl oligo(ethylene glycol)) methacrylate; MEONP] segment bearing an active ester group to react with an amino compound, and (3) a cytocompatible poly[(2-methacryloyloxyethyl phosphorylcholine; MPC) segment. Moreover, hydrophobic and carboxyl groups were attached as terminals of the polymer chain. The triblock-type polymer was attached to the QD surface through a hydrophobic layer, which was covered with the QD by hydrophobic interaction. This produced hybrid QD particles (QD/MPC polymer nanoparticles). The QD/MPC polymer nanoparticles had good water-dispersion ability after the modification. A fluorescence resonance energy transfer (FRET) phenomenon between QD and fluorescence dye (Alexa) was clearly observed at pH 7.4 and 9.0 when a fluorescence dye was reacted with the poly(MEONP) segment of the polymer. However, the efficiency decreased at pH 5.0. This was due to a change in the distance between the QD and the fluorescence dye in response to the protonation degree of the poly(DEAEMA) segment. The permeability of QD/MPC polymer nanoparticles through the cell membrane was enhanced by reacting the cell-penetrating peptide, octaarginine (R8), to the carboxyl group at the end of the polymer. The R8-QD/MPC polymer/Alexa nanoparticles attached onto the HeLa cell membrane surface within 15 min after they were added to the cell culture. This attachment initiated nanoparticle penetration of the cell membrane by endocytosis. The nanoparticles could be followed continuously as they moved in the cell culture. The change in the FRET index was determined during this process. Use of the R8-QD/MPC polymer/Alexa nanoparticle enabled us to determine nanoparticle location, based on the surrounding local pH. We concluded that QDs, modified with a cytocompatible and pH-responsible MPC polymer, provide a new imaging and transport tool in cell-based science and engineering.

Cytocompatible and Multifunctional Polymeric Nanoparticles for Transportation of Bioactive Molecules into and Within Cells

Science and Technology of Advanced Materials. 2016  |  Pubmed ID: 27877883

Multifunctional polymeric nanoparticles are materials with great potential for a wide range of biomedical applications. For progression in this area of research, unfavorable interactions of these nanoparticles with proteins and cells must be avoided in biological environments, for example, through treatment of the nanoparticle surfaces. Construction of an artificial cell membrane structure based on polymers bearing the zwitterionic phosphorylcholine group can prevent biological reactions at the surface effectively. In addition, certain bioactive molecules can be immobilized on the surface of the polymer to generate enough affinity to capture target biomolecules. Furthermore, entrapment of inorganic nanoparticles inside polymeric matrices enhances the nanoparticle functionality significantly. This review summarizes the preparation and characterization of cytocompatible and multifunctional polymeric nanoparticles; it analyzes the efficiency of their fluorescence function, the nature of the artificial cell membrane structure, and their performance as in-cell devices; and finally, it evaluates both their chemical reactivity and effects in cells.

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