Polypeptide-Grafted Macroporous PolyHIPE by Surface-Initiated <i>N</i>-Carboxyanhydride (NCA) Polymerization as a Platform for Bioconjugation

A new class of functional macroporous monoliths from polymerized high internal phase emulsion (polyHIPE) with tunable surface functional groups was developed by direct polypeptide surface grafting. In the first step, amino-functional polyHIPEs were obtained by the addition of 4-vinylbenzyl or 4-vinylbenzylphthalimide to the styrenic emulsion and thermal radical polymerization. The obtained monoliths present the expected open-cell morphology and a high surface area. The incorporated amino group was successfully utilized to initiate the ring-opening polymerization of benzyl-l-glutamate <i>N</i>-carboxyanhydride (BLG NCA) and benzyloxycarbonyl-l-lysine (Lys­(Z)) NCA, which resulted in a dense homogeneous coating of polypeptides throughout the internal polyHIPE surfaces as confirmed by SEM and FTIR analysis. The amount of polypeptide grafted to the polyHIPE surfaces could be modulated by varying the initial ratio of amino acid NCA to amino-functional polyHIPE. Subsequent removal of the polypeptide protecting groups yielded highly functional polyHIPE-<i>g</i>-poly­(glutamic acid) and polyHIPE-<i>g</i>-poly­(lysine). Both types of polypeptide-grafted monoliths responded to pH by changes in their hydrohilicity. The possibility to use the high density of function (−COOH or −NH₂) for secondary reaction was demonstrated by the successful bioconjugation of enhanced green fluorescent protein (eGFP) and fluorescein isocyanate (FITC) on the polymer 3D-scaffold surface. The amount of eGFP and FITC conjugated to the polypeptide-grafted polyHIPE was significantly higher than to the amino-functional polyHIPE, signifying the advantage of polypeptide grafting to achieve highly functional polyHIPEs

Detection of <i>“B. hampsonii”</i> strain 30446 in pre-inoculation screening of fecal samples during the acclimation period¹.

1<p>Quantitative PCR results for all pigs at ten, seven and five days prior to inoculation in trial 1, and eight, five and two days prior to inoculation, and at inoculation in trial 2. Hyphens indicate that strain 30446 was not detected, DNQ indicates detectable but not quantifiable concentrations, and a number indicates the concentration of strain 30446 in genome equivalents/g of feces.</p

Comparison of histological and gross lesions and spirochete numbers on Warthin-Faulkner stained colonic sections¹.

1<p>Statistical comparisons were among CTRL (n = 6), INOC with mucohaemorrhagic diarrhea (n = 9) and INOC without mucohaemorrhagic diarrhea (n = 3) in trial 1. In trial 2, comparisons were among CTRL (n = 6), INOC with mucohaemorrhagic diarrhea (n = 8) and INOC without mucohaemorrhagic diarrhea (n = 4) in trial 2.</p>† ‡ #<p>*Within column, animals with similar superscripts are statistically different (<i>P</i>≤0.05).</p

Phylogenetic tree of <i>Brachyspira</i> spp.

<p>Phylogenetic tree based on alignment of 810 bp of the <i>nox</i> gene of <i>Brachyspira</i> spp., including <i>“B. hampsonii”</i> strain 30446. The alignment was created using CLUSTALw, followed by distance calculation (F84 matrix) and neighbour joining using PHYLIP. The tree is a consensus of 100 boostrap iterations, and bootstrap values are indicated at the major nodes. GenBank accession numbers for <i>nox</i> sequences are indicated in the tree. Scale bar indicates 0.02 substitutions per site.</p

Perianal fecal staining.

<p>In some pigs, mucohaemorrhagic feces adheres to the perianal region of pigs following inoculation with pure broth <i>“Brachyspira hampsonii”</i> strain 30446. Both images are taken on day 8 PI: frank blood with a clot (A), blood with mucus (B).</p

Fecal consistency, shedding and tissue concentrations in pigs following inoculation with tissue homogenate containing <i>“Brachyspira hampsonii”</i> strain 30446.

<p>Daily fecal consistency scores (line, left ordinate; 0 = formed, normal; 1 = soft, wet cement consistency; 2 = runny or watery; 3 = mucoid diarrhea; or 4 = bloody diarrhea). Fecal smear spirochete scores (grey bars, left ordinate: 0 = negative; 1 = less than 1 spirochete/high power field (hpf); 2 = between 2 and 10 spirochetes/hpf; 3 = between 11 and 49 spirochetes/hpf; 4 = greater than 50 spirochetes/hpf). Strain 30446 DNA concentration (copies/g, triangles, right ordinate) in colon tissue samples collected at necropsy. Pig IDs are indicated in the upper right corner of each panel. ND = fecal smear spirochete score not done.</p

<i>“Brachyspira hampsonii”</i> strain 30446 in Warthin-Faulkner stained colonic sections.

<p>Histologic sections of spiral colon taken from pigs with mucohaemorrhagic diarrhea examined with Warthin-Faulkner silver staining. Spirochetes can be seen in the intestinal crypts (A) and along the epithelial surface (B).</p

Colonic mucosal lesions associated with <i>“Brachyspira hampsonii”</i> strain 30446.

<p>Gross colonic mucosal lesions observed following inoculation with pure broth culture containing strain 30446. Images are of different INOC pigs euthanized between D6 and D12 post-inoculation. A = mild to moderate hyperemia and/or congestion with scant mucous deposited on mucosal surface; B = moderate to severe mucosal congestion with normal looking contents adhering to mucosa; C = moderate to severe fibrinomucoid exudate adhering to hyperemic mucosal surface prior to washing; D = patchy fibrinomucoid exudate adhering to hyperemic mucosal surface after washing; E = severe fibrinonecrotic colitis; F = thick adherent mucoid exudate on mucosal surface of colon.</p

Fecal consistency, shedding and tissue concentrations in pigs following inoculation with pure broth cultivated <i>“Brachyspira hampsonii”</i> strain 30446.

<p>Fecal consistency scores (line, left ordinate: 0 = formed, normal; 1 = soft, wet cement consistency; 2 = runny or watery; 3 = mucoid diarrhea; or 4 = bloody diarrhea). Fecal smear spirochete scores (grey bars, left ordinate: 0 = less than 1 spirochete/high power field (hpf); 1 = between 2 and 10 spirochetes/hpf; 2 = between 11 and 49 spirochetes/hpf; 3 = greater than 50 spirochetes/hpf). Strain 30446 DNA concentration in feces (triangles, right ordinate), upside down triangles indicate DNQ. The red bar at the top of each panel indicates the semi-quantitative fecal culture score (0 = negative; 1 = less than 10 colonies/1° streak; 2 = less than 10 colonies/2° streak; 3 = less than 10 colonies/3°streak; 4 = less than 10 colonies/4° streak). Pig IDs are indicated in the upper right corner of each panel. ND = fecal smear spirochete score not done.</p