Polymers for binding of the gram-positive oral pathogen <i>Streptococcus mutans</i> - Fig 3

<p>Binding of coumarin 343-tagged a) cationic <b>(3)_25-100%</b> and b) sulfobetaine <b>(4)_25-100%</b> polymers at different degrees of functionalization, to <i>E</i>. <i>coli</i> and <i>S</i>. <i>mutans</i> in bacterial suspensions of OD₆₀₀ 0.1, and 1.0 mg mL^-1 polymer solutions. Area of fluorescence (%) was quantified using ImageJ. Error bars represent standard deviations on independent experiments (N = 3). Fluorescence micrographs are shown for fully functionalised (c, d) cationic and (e, f) sulfobetaine polymers, <b>(3)</b>_<b>100%</b> and <b>(4)</b>_<b>100%</b>, respectively using the 488 nm (green) channel.</p

Selective polymer binding in mixed bacterial cultures: schematic representation of bacteria cross-over experiment.

<p>a) Bacteria with an OD₆₀₀ = 0.1 were mixed with 1.0 mg mL^-1 of polymer in deionized water and incubated for 30 minutes before washing with PBS three times, re-suspending in PBS and mounting 10 μL for microscopic imaging. b) and c) Overlapped fluorescence microscopy images of mixed culture experiment. Images recorded sequentially, then overlapped. Experiments were performed in duplicate (b) and c)).</p

Polymers for binding of the gram-positive oral pathogen <i>Streptococcus mutans</i> - Fig 4

<p>(A) Binding of coumarin 343-tagged sulfobetaine polymer <b>(4)</b>_<b>100%</b> to <i>E</i>. <i>coli</i>, <i>S</i>. <i>mutans</i>, <i>V</i>. <i>Harveyi</i> and <i>S</i>. <i>Aureus</i> in bacterial suspensions of OD₆₀₀ 0.1, and 1.0 mg mL^-1 polymer solutions (scale bars = 5 μm). Representative fluorescence micrographs are shown using the green channel (488 nm excitation). Area of fluorescence (%) was quantified using ImageJ. Error bars represent standard deviations of three equivalent areas on three different micrographs. (B) Bacterial aggregation mediated by sulfobetaine polymer <b>(4)</b>_<b>100%</b>, as quantified <i>via</i> master sizer (Coulter counter) analysis of polymer—bacteria clusters.</p

インド デ ヌノ オ サワル

<p>(A) Binding of coumarin 343-tagged sulfobetaine polymer <b>(4)</b>_<b>100%</b> to <i>E</i>. <i>coli</i>, <i>S</i>. <i>mutans</i>, <i>V</i>. <i>Harveyi</i> and <i>S</i>. <i>Aureus</i> in bacterial suspensions of OD₆₀₀ 0.1, and 1.0 mg mL^-1 polymer solutions (scale bars = 5 μm). Representative fluorescence micrographs are shown using the green channel (488 nm excitation). Area of fluorescence (%) was quantified using ImageJ. Error bars represent standard deviations of three equivalent areas on three different micrographs. (B) Bacterial aggregation mediated by sulfobetaine polymer <b>(4)</b>_<b>100%</b>, as quantified <i>via</i> master sizer (Coulter counter) analysis of polymer—bacteria clusters.</p

Mannosylated (5)_Man and galactosylated (5)_Gal glycopolymers used in this study.

<p>Mannosylated (5)_Man and galactosylated (5)_Gal glycopolymers used in this study.</p

Metabolic activity of Caco-2 cell line incubated with (red squares ■) galactosylated (5)_Gal or (green circles ●) mannosylated (5)_Man glycopolymers for 24 hours in a concentration range 0.010–5.0 mg mL^-1 for 24 h.

<p>Cell viability is expressed as a percentage of MTT metabolized by a control group of untreated cells. Experiments were performed in triplicate.</p

Experimental degrees of quaternization and sulfobetainization of fluorescent pDMAEMA (2).

<p>Experimental degrees of quaternization and sulfobetainization of fluorescent pDMAEMA (2).</p

Generation of fluorescent quaternized pDMAEMA libraries (3) and (4).

<p><i>Reagents and conditions</i>: i) CuBr, (E)-<i>N</i>-(pyridine-2-ylmethylene)propan-1-amine, 70°C toluene; ii) Coumarin 343 2’-bromoethyl ester, potassium iodide, acetone, 45 to 55°C. iii) THF, methyl iodide. Initial (polymer tertiary amino groups): (methyl iodide): 1:0.25, 1:0.5, 1:0.75, 1:1 mol:mol, iv) THF, 1,3-propane sultone. Initial [polymer tertiary amino groups]₀: [1,3-propane sultone]₀: 1:0.25, 1:0.5, 1:0.75, 1:1 mol:mol.</p

Polymers for binding of the gram-positive oral pathogen <i>Streptococcus mutans</i>

<div><p><i>Streptococcus mutans</i> is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind <i>S</i>. <i>mutans</i> and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either <i>N</i>,<i>N</i>,<i>N</i>-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for <i>S</i>. <i>mutans</i> NCTC 10449, whilst <i>Escherichia coli</i> MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for <i>S</i>. <i>mutans</i>, in both isolated and <i>S</i>. <i>mutans</i>–<i>E</i>. <i>coli</i> mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive <i>S</i>. <i>mutans</i> and <i>S</i>. <i>aureus</i> compared to Gram negative <i>E</i>. <i>coli</i> and <i>V</i>. <i>harveyi</i>. A key initial stage of <i>S</i>. <i>mutans</i> pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and β-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either <i>S</i>. <i>mutans</i> or <i>E</i>. <i>coli</i> can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure–property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive <i>S</i>. <i>mutans</i> could be achieved by fine-tuning of the recognition elements in the polymer ligands.</p></div