Biocompatibility and swelling ratio of OPF/SMA hydrogel discs.

<p>Cell viability of MC3T3-E1 (A) and W20-17 (B) cells incubated with OPF/SMA copolymer hydrogel films. Cell viability determined after 72 hour incubation in the presence of OPF/SMA hydrogel discs. Viability assessed with CellTiter Glo 96 MTS viability assay (ProMega). Hydrogels were directly placed into the dishes and were in contact with cell cultures during the incubation time. Hydrogel discs (as described) were swollen in either, double distilled water, DPBS, or a solution of vancomycin hydrochloride (400 μg/mL) in double distilled water. The hydrogels were dried, weighed, then swollen again and weighed again. Swelling ratio (SR) calculated as described in Materials and Methods. Error bars represent +/- one standard deviation, N = 3 in all groups. (*) indicates a statistically significant difference (p<0.05) between DPBS and double distilled water groups.</p

Immunohistochemistry Analysis of Tumors from the PBS and ACG44 groups.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4A and 4B</a> show representative images of H&E and Ki-67 stained tumor tissues, respectively, from the PBS treated group whereas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4C and 4D</a> show images of H&E and Ki-67 staining of tumor tissue from the ACG44 treated group. All images were taken with 20× magnification. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4E</a> is quantification of the Ki-67 positive proliferative nuclei shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figures 4B and 4D</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4F</a> is a tumor image of Ki-67 staining from the ACG44 treated group, taken at 100 X to show gold accumulation (black spots) at a high magnification.</p

Role of pre-incubation with serum, C225 and NBMPR on targeting efficacy of gold nanoconjugates and their <i>in vitro</i> biological function.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2A</a> depicts the absorbance spectrums of GNP, AC4 and ACG44 before and after pre-incubation with serum either 15 minutes at room temperature or 2 hrs at 37°C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2B and 2D</a> are transmission electron microscopy images of the <i>in vitro</i> uptake of ACG44 and AIG44 in AsPC-1 cells, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2C</a> describes the effect of pre-incubation with serum on the cellular uptake of the nanoconjugates into AsPC-1 cells analyzed for gold content utilizing INAA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2E</a> also depicts INAA analysis of cellular uptake of ACG44 and AIG44 in AsPC-1 cells, both with/without pre-incubation with C225 or NBMPR to demonstrate possible uptake mechanisms. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2F</a> shows the anti-proliferative effect of ACG44, AIG44 and CG44 on AsPC-1 cells determined through ³H-thymidine incorporation.</p

Analysis of vancomycin loading in OPF/SMA hydrogels.

<p>(A) OPF/SMA 40% hydrogels were incubated with vancomycin at 400 μg/mL for 1 hour, 4 hours, 8 hours, 24 hours, or 72 hours. The concentrations of vancomycin in solution were then measured with HPLC coupled to UV-Vis detection at 280 nm and drug loading was determined by subtracting the initial concentration of drug from the final concentration after completion of vancomycin loading. (B) Hydrogels were incubated for 24 hours, samples were collected, and the concentration of vancomycin in solution was determined as described. Amount of drug loaded was calculated and loading efficiency was determined by dividing the mass of loaded drug by the dried hydrogel mass, such that loading efficiency is represented as μg vancomycin per mg hydrogel. (C) Maximal drug loading was determined by incubating OPF/SMA 30% hydrogel samples in increasingly concentrated solutions of vancomycin in double distilled water for 24 hours. Loading efficiency (Eff_l) was determined by measurement of the final drug concentration in the distilled water (C_f) after completion of the loading cycle, followed by comparison to concentration of initial solution (C_i) via the equation (Eff_l) = {[(C_i)—(C_f)] / (C_i)}*<b>100</b>%. (D) Diagram representing drug loading experiment. In all experiments, concentrations of vancomycin remaining were determined from a standard curve of vancomycin solution that was incubated under identical conditions to the sample of interest. Error bars represent +/- one standard deviation, N = 3 in all groups. Statistical representation: (*) indicates p<0.05.</p

Schematic depicting synthesis of OPF/SMA hydrogels.

<p>OPF/SMA hydrogels synthesized as described. Idealized OPF chain represented in blue, SMA is depicted in red, crosslinked between OPF chains. Vancomycin shown as red stars within the hydrogel.</p