Functional stability of GM1-NPs in the presence of intestinal luminal factors.

<p>(A) Intensity-weighted size distribution of GM1-NPs incubated 30 min in distilled water or diluted porcine bile solution (1:16 dilution in water). (B) Fluorescence imaging of DiD-labeled GM1-NPs absorbed with FITC-CTB and incubated for 30 min in 1:16 diluted porcine bile solution. (C) GM1-NPs were loaded with FITC-CTB for 30 min, washed, and resuspended in luminal fluid from the small intestine of normal adult mice, or PBS as a control. After incubation for 24 h at 37°C, particle-bound and free FITC-CTB were separated by dialysis, and bound FITC-CTB was measured by fluorescence spectroscopy and related to the initial amount bound (mean ± SD, n = 3). Background readings were obtained with free FITC-CTB without GM1-NPs. (D) Fecal homogenates from mice were mixed 1:1 with GM1-NPs or PEG-NPs in culture media, and incubated for 1 h at 37°C, after which CT (10 ng/mL) was added for an additional 1 h before addition to HCA7 monolayers. After 2 h, cAMP levels in the supernatants were determined by ELISA (mean ± SD, n = 3; *p<0.05 vs PEG-NPs).</p

<i>In vivo</i> efficacy of GM1-NPs against CT and live <i>V</i>. <i>cholerae</i>.

<p>(A) Ligated intestinal loops were prepared in the distal small intestine of adult C57BL/6 mice, and injected with PBS as a control, or with 2.5 μg CT, without and with prior addition of GM1-NPs or control PEG-NPs. Fluid accumulation in the loops was determined after 4 h, and related to loop length (each point represents one animal, horizontal lines are geometric means; *p<0.05 vs PEG-NPs). (B) Loops were injected with PBS as a control, or live <i>V</i>. <i>cholerae</i> with GM1-NPs or control PEG-NPs. Fluid accumulation was determined after 16 h (each point represents one animal, horizontal lines are geometric means; *p<0.05 vs PEG-NPs). (C) Images of representative intestinal loops. (D) cAMP was measured in the luminal fluid collected from loops after injection of live <i>V</i>. <i>cholera</i>e with GM1-NPs or control PEG-NPs (n = 3; mean ± SD; *p<0.05 vs PEG-NPs).</p

Preparation and physical characterization of GM1-coated nanoparticles.

<p>(A) Schematic of GM1-NP fabrication. Poly(lactic-co-glycolic acid) (PLGA) dissolved in acetonitrile (CH₃CN) is added to an aqueous solution containing GM1. After acetonitrile evaporation, nanoparticles with a polymeric core and a lipid shell are formed. (B) Intensity-weighted size distribution of representative preparations of GM1-NPs and control PEG-NPs, and PLGA-NPs with a PLGA core but without a lipid shell. (C) Zeta potential of the indicated nanoparticle preparations (n = 3; mean ± SD; *p<0.05 vs. PLGA-NPs). (D) Nanoparticle size measurements over two weeks of incubation in distilled water or PBS (n = 3; mean ± SD). (E) Transmission electron micrograph of GM1-NPs.</p

Model of therapeutic effect of GM1-NPs.

<p>GM1-coated nanoparticles act as decoys to absorb CT produced by <i>V</i>. <i>cholerae</i> before it can bind to epithelial cells to stimulate cAMP production and epithelial chloride secretion, and inhibit sodium absorption.</p

Neutralization of CT activity with GM1-NPs.

<p>(A) A fixed concentration (10 ng/mL) of CT was combined with increasing amounts of the indicated nanoparticles, and the mixtures were added to confluent monolayers of human HCA7 intestinal epithelial cells. After 2 h, levels of secreted cAMP were determined in the supernatants by ELISA (n = 3; mean ± SD). (B) A fixed amount (1 μg/mL) of the indicated nanoparticles were combined with increasing concentrations of CT, the mixtures were added to HCA7 monolayers for 2 h, and levels of secreted cAMP levels were measured by ELISA (n = 3; mean ± SD). (C) GM1-NPs or control PEG-NPs were added to HCA7 monolayers, which were then infected for 2 h with live <i>V</i>. <i>cholerae</i> or left uninfected, and secreted cAMP was determined (n = 3; mean ± SD; *p<0.05 vs PEG-NPs).</p