Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.     

Gbetagamma activation of Src induces caveolae-mediated endocytosis in endothelial cells

Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to G(i)-induced Src kinase activation, which in turn signals dynamin-2-mediated fission and directed migration of caveolae-derived vesicles from apical to basal membrane. In this study, we investigated the possible role of the Gbetagamma heterodimer in signaling G(i)-induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of beta-adrenergic receptor kinase (ct-betaARK), an inhibitor Gbetagamma signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ct-betaARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of Gbetagamma using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ct-betaARK. Taken together, our results demonstrate that Gbetagamma activation of Src signals caveolae-mediated endocytosis and transendothelial albumin transport via transcytosis.     

Cellular internalization of poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymer micelles

Poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) block copolymers self-assemble into micelles in aqueous solution. We have examined whether these micelles can internalize into P19 cells in vitro. Fluorescently labeled PEO(45)-b-PCL(23) block copolymer was prepared by conjugating a tetramethylrhodamine molecule to the end of the hydrophobic PCL block. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies yielded 24 +/- 2 and 25 +/- 2 nm, respectively, for the diameters of the micelles. The studies also showed that chemical labeling did not effect the morphology or size. When the rhodamine-labeled PEO(45)-b-PCL(23) block copolymer micelles were tested in vitro, time-, concentration-, and pH-dependence of the internalization process suggested that internalization proceeded by endocytosis. The results from these studies provide the first direct evidence for the internalization of PEO(45)-b-PCL(23) micelles. Future studies will utilize multiple labeling of these micelles, allowing questions to be addressed related to the fate of internalized micelles as drug carriers, the destination of the incorporated drugs or fluorescent probes released from micelles, and the identification of the subcellular localization of the whole drug-carrier system within cells, both in vitro and in vivo.     

Endocytosis via caveolae

Caveolae are flask-shaped invaginations present in the plasma membrane of many cell types. They have long been implicated in endocytosis, transcytosis, and cell signaling. Recent work has confirmed that caveolae are directly involved in the internalization of membrane components (glycosphingolipids and glycosylphosphatidylinositol-anchored proteins), extracellular ligands (folic acid, albumin, autocrine motility factor), bacterial toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus). Unlike clathrin-mediated endocytosis, internalization through caveolae is a triggered event that involves complex signaling. The mechanism of internalization and the subsequent intracellular pathways that the internalized substances take are starting to emerge.     

Synthesis, characterization, antitumor activity of pluronic mimicking copolymer micelles conjugated with doxorubicin via acid-cleavable linkage

Pluronic mimicking poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer having multiple hydroxyl groups in the PPO middle segment (core-functionalized Pluronic: CF-PLU) was synthesized for conjugation of doxorubicin (DOX). DOX was conjugated on the multiple hydroxyl groups of CF-PLU via an acid-labile hydrazone linkage (CF-PLU-DOX). In aqueous solution, CF-PLU-DOX copolymers self-assembled to form a core/shell-type micelle structure consisting of a hydrophobic DOX-conjugated PPO core and a hydrophilic PEO shell layer. The conjugated DOX from CF-PLU-DOX micelles was released out more rapidly at pH 5 than pH 7.4, indicating that the hydrazone linkage was cleaved under acidic condition. CF-PLU-DOX micelles exhibited greatly enhanced cytotoxicity for MCF-7 human breast cancer cells compared to naked DOX, while CF-PLU copolymer itself showed extremely low cytotoxicity. Flow cytometry analysis revealed that the extent of cellular uptake for CF-PLU-DOX micelles was greater than free DOX. Confocal image analysis also showed that CF-PLU-DOX micelles had a quite different intracellular distribution profile from free DOX. CF-PLU-DOX micelles were mainly distributed in the cytoplasm, endosomal/lysosomal vesicles, and nucleus, while free DOX was localized mainly within the nucleus, suggesting that CF-PLU-DOX micellar formulation might be advantageously used for overcoming the multidrug resistance (MDR) effect, which gradually develops in many tumor cells during repeated drug administration.     

Synthesis and aqueous solution properties of novel sugar methacrylate-based homopolymers and block copolymers

We report the facile preparation of a range of novel, well-defined cyclic sugar methacrylate-based polymers without recourse to protecting group chemistry. 2-Gluconamidoethyl methacrylate (GAMA) and 2-lactobionamidoethyl methacrylate (LAMA) were prepared directly by reacting 2-aminoethyl methacrylate with D-gluconolactone and lactobionolactone, respectively. Homopolymerization of GAMA and LAMA by atom transfer radical polymerization (ATRP) gave reasonably low polydispersities as judged by aqueous gel permeation chromatography. A wide range of sugar-based block copolymers were prepared using near-monodisperse macroinitiators based on poly(ethylene oxide) [PEO], poly(propylene oxide) [PPO], or poly(e-caprolactone) [PCL] and/or by sequential monomer addition of other methacrylic monomers such as 2-(diethylamino)ethyl methacrylate [DEA], 2-(diisopropylaminoethyl methacrylate [DPA], or glycerol monomethacrylate [GMA]. The reversible micellar self-assembly of selected sugar-based block copolymers [PEO23-GAMA50-DEA100, PEO23-LAMA30-DEA50, PPO33-GAMA50, and PPO33-LAMA50] was studied in aqueous solution as a function of pH and temperature using dynamic light scattering, transmission electron microscopy, surface tensiometry, and 1H NMR spectroscopy.     

Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis

Influenza virus has been described to enter host cells via clathrin-mediated endocytosis. However, it has also been suggested that other endocytic routes may provide additional entry pathways. Here we show that influenza virus may enter and infect HeLa cells that are unable to take up ligands by clathrin-mediated endocytosis. By overexpressing a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis, we demonstrate that while transferrin uptake and Semliki Forest virus infection were prevented, influenza virus could enter and infect cells expressing Eps15Delta95/295. This finding is supported by the successful infection of cells with influenza virus in the presence of chemical treatments that block endocytosis, namely, chlorpromazine and potassium depletion. We show also that influenza virus may infect cells incapable of uptake by caveolae. Treatment with the inhibitors nystatin, methyl-beta-cyclodextrin, and genistein, as well as transfection of cells with dominant-negative caveolin-1, had no effect on influenza virus infection. By combining inhibitory methods to block both clathrin-mediated endocytosis and uptake by caveolae in the same cell, we demonstrate that influenza virus may infect cells by an additional non-clathrin-dependent, non-caveola-dependent endocytic pathway. We believe this to be the first conclusive analysis of virus entry via such a non-clathrin-dependent pathway, in addition to the traditional clathrin-dependent route.     

Dynamin-mediated Internalization of Caveolae

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.     

Different Contributions of Clathrin- and Caveolae-Mediated Endocytosis of Vascular Endothelial Cadherin to Lipopolysaccharide-Induced Vascular Hyperpermeability

Vascular hyperpermeability induced by lipopolysaccharide (LPS) is a common pathogenic process in cases of severe trauma and sepsis. Vascular endothelial cadherin (VE-cad) is a key regulatory molecule involved in this process, although the detailed mechanism through which this molecule acts remains unclear. We assessed the role of clathrin-mediated and caveolae-mediated endocytosis of VE-cad in LPS-induced vascular hyperpermeability in the human vascular endothelial cell line CRL-2922 and determined that vascular permeability and VE-cad localization at the plasma membrane were negatively correlated after LPS treatment. Additionally, the loss of VE-cad at the plasma membrane was caused by both clathrin-mediated and caveolae-mediated endocytosis. Clathrin-mediated endocytosis was dominant early after LPS treatment, and caveolae-mediated endocytosis was dominant hours after LPS treatment. The caveolae-mediated endocytosis of VE-cad was activated through the LPS-Toll-like receptor 4 (TLR4)-Src signaling pathway. Structural changes in the actin cytoskeleton, specifically from polymerization to depolymerization, were important reasons for the switching of the VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated. Our findings suggest that clathrin-mediated and caveolae-mediated endocytosis of VE-cad contribute to LPS-induced vascular hyperpermeability, although they contribute via different mechanism. The predominant means of endocytosis depends on the time since LPS treatment.     

Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells

Albumin transcytosis, a determinant of transendothelial permeability, is mediated by the release of caveolae from the plasma membrane. We addressed the role of Src phosphorylation of the GTPase dynamin-2 in the mechanism of caveolae release and albumin transport. Studies were made in microvascular endothelial cells in which the uptake of cholera toxin subunit B, a marker of caveolae, and (125)I-albumin was used to assess caveolae-mediated endocytosis. Albumin binding to the 60-kDa cell surface albumin-binding protein, gp60, induced Src activation (phosphorylation on Tyr(416)) within 1 min and resulted in Src-dependent tyrosine phosphorylation of dynamin-2, which increased its association with caveolin-1, the caveolae scaffold protein. Expression of kinase-defective Src mutant interfered with the association between dynamin-2, which caveolin-1 and prevented the uptake of albumin. Expression of non-Src-phosphorylatable dynamin (Y231F/Y597F) resulted in reduced association with caveolin-1, and in contrast to WT-dynamin-2, the mutant failed to translocate to the caveolin-rich membrane fraction. The Y231F/Y597F dynamin-2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport. Thus, Src-mediated phosphorylation of dynamin-2 is an essential requirement for scission of caveolae and the resultant transendothelial transport of albumin.     

Polycationic block copolymers of poly(ethylene oxide) and poly(propylene oxide) for cell transfection

A facile, one-step synthesis of cationic block copolymers of poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and copolymers of poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO) has been developed. The PEO-PPO-PEO-pDMAEMA (L92-pDMAEMA) and PEO-pDMAEMA copolymers were obtained via free radical polymerization of DMAEMA initiated by polyether radicals generated by cerium(IV). Over 95% of the copolymer fraction was of molecular mass ranging from 6.9 to 7.1 kDa in size, indicating the prevalence of the polyether-monoradical initiation mechanism. The L92-pDMAEMA copolymers possess parent surfactant-like surface activity. In contrast, the PEO-pDMAEMA copolymers lack significant surface activity. Both copolymers can complex with DNA. Hydrodynamic radii of the complexes of the L92-pDMAEMA and PEO-pDMAEMA with plasmid DNA ranged in size from 60 to 400 nm, depending on the copolymer/DNA ratio. Addition of Pluronic P123 to the L92-pDMAEMA complexes with DNA masked charges and decreased the tendency of the complex to aggregate, even at stoichiometric polycation/DNA ratios. The transfection efficiency of the L92-pDMAEMA copolymer was by far greater than that of the PEO-pDMAEMA copolymer. An extra added Pluronic P123 further increased the transfecton efficacy of L92-pDMAEMA, but did not affect that of PEO-pDMAEMA.     

The adipocyte plasma membrane caveolin functional/structural organization is necessary for the efficient endocytosis of GLUT4

It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-beta-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin- and clathrin-dependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced ( approximately 40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by approximately 20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.     

Distinct role of clathrin-mediated endocytosis in the functional uptake of cholera toxin

The involvement of the clathrin-mediated endocytic internalization route in the uptake of cholera toxin (CT) was investigated using different cell lines, including the human intestinal Caco-2 and T84 cell lines, green monkey Vero cells, SH-SY5Y neuroblastoma cells and Madin-Darby canine kidney cells. Suppression of the clathrin-mediated endocytic pathway by classical biochemical procedures, like intracellular acidification and potassium depletion, inhibited cholera toxin uptake by up to about 50% as well as its ability to raise intracellular levels of cAMP. Also prior exposure of these cell types to the cationic amphiphilic drug chlorpromazine reduced the functional uptake of cholera toxin, even to a greater extent. These effects were dose- and cell type-dependent, suggesting an involvement of clathrin-mediated endocytosis in the functional uptake of cholera toxin. For a more straightforward approach to study the role of the clathrin-mediated uptake in the internalization of cholera toxin, a Caco-2(eps-) cell line was exploited. These Caco-2(eps-) cells constitutively suppress the expression of epsin, an essential accessory protein of clathrin-mediated endocytosis, thereby selectively blocking this internalization route. CT uptake was found to be reduced by over 60% in Caco-2(eps-) paralleled by a diminished ability of CT to raise the level of cAMP. The data presented suggest that the clathrin-mediated uptake route fulfils an important role in the functional internalization of cholera toxin in several cell types.     

Clathrin-Dependent and Clathrin-Independent Endocytosis are Differentially Sensitive to Insertion of Poly (Ethylene Glycol)-Derivatized Cholesterol in the Plasma Membrane

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036–3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.     

Electron density mapping of triblock copolymers associated with model biomembranes: insights into conformational states and effect on bilayer structure

One-dimensional electron-density profiles derived from synchrotron small-angle X-ray scattering (SAXS) have been constructed and used to determine the conformational state of selected poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers and the region of their association with a lipid bilayer. The number of molecular repeat units in the hydrophobic PPO block has been found to determine both the nature of triblock polymer-membrane association and the conformational state of the symmetric, flanking hydrophilic PEO units. For DMPC-based biomembranes, polymers whose PPO chain length is less than that of the bilayer thickness insert weakly into the membrane with the PEO blocks on the same side of the bilayer, leading to delocalization of the PEO at the membrane-water interface. This polymer architecture has been found not to alter the membrane fluidity and roughness. Conversely, polymers whose chain length is sufficient to span the lipid bilayer are tightly integrated, projecting their PEO chains into the water channels on opposing sides of the bilayer. The coiled conformational state of the PEO chains produces steric pressure on the bilayer, causing a thinning of the membrane and leading to a rigid, less-mobile bilayer than systems where the polymer is introduced as the lipid conjugate.     

Bovine papillomavirus type 1: from clathrin to caveolin

Viruses may infect cells through clathrin-dependent, caveolin-dependent, or clathrin- and caveolin-independent endocytosis. Bovine papillomavirus type 1 (BPV1) entry into cells has been shown to occur by clathrin-dependent endocytosis, a pathway that involves the formation of clathrin-coated pits and fusion to early endosomes. Recently, it has been demonstrated that the closely related JC virus can enter cells in clathrin-coated vesicles and subsequently traffic to caveolae, the organelle where vesicles of the caveolin-dependent pathway deliver their cargo. In this study, we use immunofluorescence staining of BPV1 pseudovirions to show that BPV1 overlaps with the endosome marker EEA1 early during infection and later colocalizes with caveolin-1. We provide evidence through the colocalization of BPV1 with transferrin and cholera toxin B that BPVl trafficking may not be restricted to the clathrin-dependent pathway. Disrupting the entry of caveolar vesicles did not affect BPV1 infection; however, we show that blocking the caveolar pathway postentry results in a loss of BPV1 infection. These data indicate that BPV1 may enter by clathrin-mediated endocytosis and then utilize the caveolar pathway for infection, a pattern of trafficking that may explain the slow kinetics of BPV1 infection.     

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell''s waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell''s waltzer fibroblasts.     

Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background

The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.

Methodology/Principal Findings

We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t_1/2<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.

Conclusion

It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.     

Adsorption of a PEO–PPO–PEO triblock copolymer on metal oxide surfaces with a view to reducing protein adsorption and further biofouling

Abstract

Biomolecule adsorption is the first stage of biofouling. The aim of this work was to reduce the adsorption of proteins on stainless steel (SS) and titanium surfaces by modifying them with a poly(ethylene oxide) (PEO)–poly(propylene oxide) (PPO)–PEO triblock copolymer. Anchoring of the central PPO block of the copolymer is known to be favoured by hydrophobic interaction with the substratum. Therefore, the surfaces of metal oxides were first modified by self-assembly of octadecylphosphonic acid. PEO–PPO–PEO preadsorbed on the hydrophobized surfaces of titanium or SS was shown to prevent the adsorption of bovine serum albumin (BSA), fibrinogen and cytochrome C, as monitored by quartz crystal microbalance (QCM). Moreover, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry were used to characterize the surfaces of the SS and titanium after competitive adsorption of PEO–PPO–PEO and BSA. The results show that the adsorption of BSA is well prevented on hydrophobized surfaces, in contrast to the surfaces of native metal oxides.     

Mesoscopic simulation studies on micellar phases of Pluronic P103 solution

The microphase separation dynamics of the triblock copolymer surfactant P103 [(ethylene oxide)₁₇(propylene oxide)₆₀(ethylene oxide)₁₇] was investigated by a dynamic variant of mean-field density functional theory. Different self-assembled aggregates, spherical micelles, micellar clusters and disk-like micelles, are explored in the solution. The spherical micelle above critical micelle concentration (CMC) is a dense core consisting mainly of PPO and a hydrated PEO swollen corona, and is in good agreement with the experimental results concerning their structures. At a concentration of 10–15%, micellar clusters with a larger PPO core form as a result of coalescence among spherical micelles. At concentrations above 16% by volume, a series of disk-like micelles come into being. The order parameters show that spherical micelles are easily formed, while the micellar clusters or disk-like micelles need a longer time to reach steady equilibrium. The results show that mesoscopic simulation can augment experimental results on amphiphilic polymers, and provide some mesoscopic information at the mesoscale level. Figure Coalescence of Micelles with time evolution for 15% vol system. □ represents spherical micelle that coalesce. (a) 180 μs, (b) 190 μs, (c)225 μs, and (d) 250 μs