Thermoresponsive Hydrogel of Diblock Methylcellulose: Formation of Ribbonlike Supramolecular Nanostructures by Self-Assembly

This article provides detailed insight into the thermoresponsive gelation mechanism of industrially produced methylcellulose (MC), highlighting the importance of diblock structure with a hydrophobic sequence of 2,3,6-tri-<i>O</i>-methyl-glucopyranosyl units for this physicochemical property. We show herein, for the first time, that well-defined diblock MC self-assembles thermoresponsively into ribbonlike nanostructures in water. A cryogenic transmission electron microscopy (cryo-TEM) technique was used to detect the ribbonlike nanostructures formed by the diblock copolymers consisting of hydrophilic glucosyl or cellobiosyl and hydrophobic 2,3,6-tri-<i>O</i>-methyl-cellulosyl blocks, methyl β-d-glucopyranosyl-(1→4)-2,3,6-tri-<i>O</i>-methyl-celluloside <b>1</b> (G-236MC, DP_<i>n</i> = 10.7, DS = 2.65), and methyl β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-2,3,6-tri-<i>O</i>-methyl-celluloside <b>2</b> (GG-236MC, DP_<i>n</i> = 28.2, DS = 2.75). Rheological measurements revealed that the gel strength of a dispersion of GG-236MC (<b>2</b>, 2.0 wt %) in water at 70 °C was 3.0 times stronger than that of commercial MC SM-8000, although the molecular weight of GG-236MC (<b>2</b>) having <i>M</i>_w = 8 × 10³ g/mol was 50 times smaller than that of SM-8000 having <i>M</i>_w = 4 × 10⁵ g/mol. Cryo-TEM observation suggested that the hydrogel formation of the diblock copolymers could be attributed to the entanglement of ribbonlike nanostructures self-assembled by the diblock copolymers in water. The cryo-TEM micrograph of GG-236MC (<b>2</b>) at 5 °C showed rectangularly shaped nanostructures having a thickness from 11 to 24 nm, although G-236MC (<b>1</b>) at 20 °C showed no distinct self-assembled nanostructures. The ribbonlike nanostructures of GG-236MC (<b>2</b>) having a length ranging from 91 to 864 nm and a thickness from 8.5 to 27.1 nm were detected above 20 °C. Small-angle X-ray scattering measurements suggested that the ribbonlike nanostructures of GG-236MC (<b>2</b>) consisted of a bilayer structure with a width of ca. 40 nm. It was likely that GG-236MC (<b>2</b>) molecules were oriented perpendicularly to the long axis of the ribbonlike nanostructure. In addition, wide-angle X-ray scattering measurements revealed that GG-236MC (<b>2</b>) in its hydrogel formed the same crystalline regions as 2,3,6-tri-<i>O</i>-methylcellulose. The influence of the DP of diblock MC with a DS of around 2.7 on the gelation behavior will be discussed

Thermoresponsive Hydrogel of Diblock Methylcellulose: Formation of Ribbonlike Supramolecular Nanostructures by Self-Assembly

This article provides detailed insight into the thermoresponsive gelation mechanism of industrially produced methylcellulose (MC), highlighting the importance of diblock structure with a hydrophobic sequence of 2,3,6-tri-<i>O</i>-methyl-glucopyranosyl units for this physicochemical property. We show herein, for the first time, that well-defined diblock MC self-assembles thermoresponsively into ribbonlike nanostructures in water. A cryogenic transmission electron microscopy (cryo-TEM) technique was used to detect the ribbonlike nanostructures formed by the diblock copolymers consisting of hydrophilic glucosyl or cellobiosyl and hydrophobic 2,3,6-tri-<i>O</i>-methyl-cellulosyl blocks, methyl β-d-glucopyranosyl-(1→4)-2,3,6-tri-<i>O</i>-methyl-celluloside <b>1</b> (G-236MC, DP_<i>n</i> = 10.7, DS = 2.65), and methyl β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-2,3,6-tri-<i>O</i>-methyl-celluloside <b>2</b> (GG-236MC, DP_<i>n</i> = 28.2, DS = 2.75). Rheological measurements revealed that the gel strength of a dispersion of GG-236MC (<b>2</b>, 2.0 wt %) in water at 70 °C was 3.0 times stronger than that of commercial MC SM-8000, although the molecular weight of GG-236MC (<b>2</b>) having <i>M</i>_w = 8 × 10³ g/mol was 50 times smaller than that of SM-8000 having <i>M</i>_w = 4 × 10⁵ g/mol. Cryo-TEM observation suggested that the hydrogel formation of the diblock copolymers could be attributed to the entanglement of ribbonlike nanostructures self-assembled by the diblock copolymers in water. The cryo-TEM micrograph of GG-236MC (<b>2</b>) at 5 °C showed rectangularly shaped nanostructures having a thickness from 11 to 24 nm, although G-236MC (<b>1</b>) at 20 °C showed no distinct self-assembled nanostructures. The ribbonlike nanostructures of GG-236MC (<b>2</b>) having a length ranging from 91 to 864 nm and a thickness from 8.5 to 27.1 nm were detected above 20 °C. Small-angle X-ray scattering measurements suggested that the ribbonlike nanostructures of GG-236MC (<b>2</b>) consisted of a bilayer structure with a width of ca. 40 nm. It was likely that GG-236MC (<b>2</b>) molecules were oriented perpendicularly to the long axis of the ribbonlike nanostructure. In addition, wide-angle X-ray scattering measurements revealed that GG-236MC (<b>2</b>) in its hydrogel formed the same crystalline regions as 2,3,6-tri-<i>O</i>-methylcellulose. The influence of the DP of diblock MC with a DS of around 2.7 on the gelation behavior will be discussed

Thermoresponsive Hydrogel of Diblock Methylcellulose: Formation of Ribbonlike Supramolecular Nanostructures by Self-Assembly

This article provides detailed insight into the thermoresponsive gelation mechanism of industrially produced methylcellulose (MC), highlighting the importance of diblock structure with a hydrophobic sequence of 2,3,6-tri-<i>O</i>-methyl-glucopyranosyl units for this physicochemical property. We show herein, for the first time, that well-defined diblock MC self-assembles thermoresponsively into ribbonlike nanostructures in water. A cryogenic transmission electron microscopy (cryo-TEM) technique was used to detect the ribbonlike nanostructures formed by the diblock copolymers consisting of hydrophilic glucosyl or cellobiosyl and hydrophobic 2,3,6-tri-<i>O</i>-methyl-cellulosyl blocks, methyl β-d-glucopyranosyl-(1→4)-2,3,6-tri-<i>O</i>-methyl-celluloside <b>1</b> (G-236MC, DP_<i>n</i> = 10.7, DS = 2.65), and methyl β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-2,3,6-tri-<i>O</i>-methyl-celluloside <b>2</b> (GG-236MC, DP_<i>n</i> = 28.2, DS = 2.75). Rheological measurements revealed that the gel strength of a dispersion of GG-236MC (<b>2</b>, 2.0 wt %) in water at 70 °C was 3.0 times stronger than that of commercial MC SM-8000, although the molecular weight of GG-236MC (<b>2</b>) having <i>M</i>_w = 8 × 10³ g/mol was 50 times smaller than that of SM-8000 having <i>M</i>_w = 4 × 10⁵ g/mol. Cryo-TEM observation suggested that the hydrogel formation of the diblock copolymers could be attributed to the entanglement of ribbonlike nanostructures self-assembled by the diblock copolymers in water. The cryo-TEM micrograph of GG-236MC (<b>2</b>) at 5 °C showed rectangularly shaped nanostructures having a thickness from 11 to 24 nm, although G-236MC (<b>1</b>) at 20 °C showed no distinct self-assembled nanostructures. The ribbonlike nanostructures of GG-236MC (<b>2</b>) having a length ranging from 91 to 864 nm and a thickness from 8.5 to 27.1 nm were detected above 20 °C. Small-angle X-ray scattering measurements suggested that the ribbonlike nanostructures of GG-236MC (<b>2</b>) consisted of a bilayer structure with a width of ca. 40 nm. It was likely that GG-236MC (<b>2</b>) molecules were oriented perpendicularly to the long axis of the ribbonlike nanostructure. In addition, wide-angle X-ray scattering measurements revealed that GG-236MC (<b>2</b>) in its hydrogel formed the same crystalline regions as 2,3,6-tri-<i>O</i>-methylcellulose. The influence of the DP of diblock MC with a DS of around 2.7 on the gelation behavior will be discussed

Functional <i>sigB</i> and <i>agr</i> operons, but not <i>sarA</i>, are indispensable for <i>S. aureus</i> to survive phagocytosis by hMDMs.

<p>Macrophages were allowed to engulf defined strains of <i>S. aureus</i> for 2h at a MOI of 25, and the intracellular survival of bacteria on consecutive days post-phagocytosis was monitored by enumeration of the CFU of cell lysates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001409#pone-0001409-g003" target="_blank">Fig. 3A</a> legend). The data shown is representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. Bars represent mean CFU value ±SD. A. 8325-4, a natural <i>rsbU</i> defective strain lacking a functional SigB. B. SH1000, a derivative of strain 8325-4 with a restored <i>rsbU</i> gene and SigB activity. C. Newman <i>sigB</i> mutant D. Newman <i>agr</i> mutant E. Newman <i>sarA</i> mutant F. Newman wild-type</p

<i>S. aureus</i> infected macrophages retained their bactericidal functions.

<p>Control hMDMs, and cells 4 days post infection with <i>S. aureus</i> strain Newman, were stimulated to phagocytose either live bacteria at a MOI of 1∶25 (1.25×10⁷ CFU) (panel A) or latex beads (panel B). Generation of reactive oxygen species (ROS) determined as the level of the mean fluorescence intensity (MFI) was measured at various time intervals. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors.</p

Internalization of <i>S. aureus</i> strain Newman does not affect hMDM viability until the plasma membrane is permeabilized.

<p>A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and <i>S. aureus</i>-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and <i>S. aureus</i> infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001409#s4" target="_blank">Materials and Methods</a> section. All scale bars = 10 µm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and ×7,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of <i>S. aureus</i> infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by <i>S. aureus</i> infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] × 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with <i>S. aureus</i>. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate.</p

An α-hemolysin mutant of <i>S. aureus</i> strain Newman is efficiently killed in phago(lyso)somes within two days of phagocytosis.

<p>Intracellular persistence of <i>S. aureus</i> was determined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001409#pone-0001409-g003" target="_blank">Fig. 3A</a>. Bars represent mean CFU values ±SD and are representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. An electron microphotograph of infected hMDMs was obtained 1 day post-phagocytosis and shows partially (white arrows) and totally degraded (black arrows) bacterial cells inside tight vacuoles. At this time point some apparently intact bacterial cells are also present (black arrowheads). Magnification: x32,500. The photograph presented is a representative of a minimum of 20 fields observed.</p

Apparently structurally normal, intact dividing and viable <i>S. aureus</i> Newman cells persist in the intracellular vacuolar compartment on four consecutive days post-phagocytosis.

<p>The photographs presented are representative of a minimum of 20 fields observed. hMDMs were allowed to engulf bacteria for 2h at a MOI of 25, and infected macrophages were fixed with Karnovsky's fixative either immediately following phagocytosis (2 h), or on consecutive days post-phagocytosis, before being processed by standard electron microscopic techniques. At any given point post-phagocytosis dividing bacterial cells were visible (arrowheads). Magnified views of selected bacteria framed on main micrographs are shown in the bottom row labeled from A' to H', respectively. A. Control, non-infected hMDM has a morphological appearance typical for that of professional phagocyte, with vesicles localized around the nucleus (N) and only few lipid droplets (arrows). Magnification: x14,000. B. Immediately post-phagocytosis (2h), the bacteria were located predominantly in very tight membranous compartments (arrows). Magnification: x50,000. C. Already 1 day post-phagocytosis the majority of <i>S. aureus</i> cells were found in clearly defined membrane-enclosed vacuoles (arrows). A partially degraded bacterium in the vacuole is framed. Magnification: x20,000. D and E. At day 2 the cells of <i>S. aureus</i> can only be found in well defined vacuoles (arrows), occasionally spacious vacuoles (asterisk) containing several bacteria. Magnification: x32,500. F. On day 3 <i>S. aureus</i> persists in vacuoles which often reveal signs of partial membrane discontinuity (arrows). Magnification: x32,500. G and H. On day 4, the majority of bacteria were found in vacuoles with profoundly damaged or fully disintegrated membranes (arrow). Magnification: x54,000 (G) and x32,000 (H).</p

After being phagocytosed by hMDM <i>S. aureus</i> strain Newman decreasingly persists intracellularly for several days until a burst of growth on day 6.

<p>A. CFU of <i>S. aureus</i> in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf <i>S. aureus</i> at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means±SD) of the 76 we performed in triplicate. B. CFU of <i>S. aureus</i> and LDH activity levels (A_{490 nm}) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of <i>S. aureus</i>-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 µm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5^th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable <i>S. aureus</i> cells are stained green while red signals represent dead bacteria and the host cell's nuclear DNA stained mainly with propidium. Scale bar = 7.5 µm. E. Specific PCR amplification of the <i>S. aureus</i> 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of <i>S. aureus</i> Newman before infection.</p

Macrophages stimulated with interferon-γ kill engulfed <i>S. aureus</i> strains: Newman (A), ATCC 25923 (B), and COL (C) more efficiently than non-stimulated cells.

<p>Cells were stimulated with recombinant human IFNγ overnight at concentrations equivalent to human therapeutic doses (100 U ml⁻¹), and then allowed to phagocytose three different strains of <i>S. aureus</i> for 2 h. Infected cultures were processed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001409#pone-0001409-g003" target="_blank">Fig. 3A</a> and live bacteria in the whole cultures (CFU) were enumerated up to 14 days postphagocytosis. Since at the longer timepoints no bacterial growth was detected (CFU = 0) these points were not presented on the graph. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDM derived from different donors. Bars represent mean CFU value ±SD. *, p<0.05; **, p<0.01; ***, p<0.001. NS-not significant.</p