12 research outputs found
Nanodiamonds facilitate killing of intracellular uropathogenic E. coli in an in vitro model of urinary tract infection pathogenesis.
About 25-44% of women will experience at least one episode of recurrent UTI and the causative agent in over 70% of UTI cases is uropathogenic Escherichia coli (UPEC). UPEC cause recurrent UTI by evading the bladder's innate immune system through internalization into the bladder epithelium where antibiotics cannot reach or be effective. Thus, it is important to develop novel therapeutics to eliminate these intracellular pathogens. Nanodiamonds (NDs) are biocompatible nanomaterials that serve as promising candidates for targeted therapeutic applications. The objective of the current study was to investigate if 6 or 25 nm NDs can kill extracellular and intracellular UPEC in infected bladder cells. We utilized the human bladder epithelial cell line, T24, and an invasive strain of UPEC that causes recurrent UTI. We found that acid-purified 6 nm NDs displayed greater antibacterial properties towards UPEC than 25 nm NDs (11.5% vs 94.2% CFU/mL at 100 μg/mL of 6 and 25 nm, respectively; P<0.001). Furthermore, 6 nm NDs were better than 25 nm NDs in reducing the number of UPEC internalized in T24 bladder cells (46.1% vs 81.1% CFU/mL at 100 μg/mL of 6 and 25 nm, respectively; P<0.01). Our studies demonstrate that 6 nm NDs interacted with T24 bladder cells in a dose-dependent manner and were internalized in 2 hours through an actin-dependent mechanism. Finally, internalization of NDs was required for reducing the number of intracellular UPEC in T24 bladder cells. These findings suggest that 6 nm NDs are promising candidates to treat recurrent UTIs
Cytotoxicity of 6 nm and 25 nm NDs in T24 cells.
<p>T24 cells were treated with 6 nm (A) or 25 nm (B) commercial (white bars) or acid-treated (black bars) NDs for 24 hours. Cytotoxic effects of NDs were evaluated by the MTT assay. Data are representative of at least three independent experiments and depicted as mean ± SEM. There were statistically significant (<i>P</i> < 0.05) decreases in the percentages of cell survival following treatment with NDs as compared to the corresponding 0 μg/mL sample as determined by 2-way ANOVA followed by Tukey’s multiple comparison’s test.</p
Purification and characterization of 6 nm and 25 nm nanodiamonds.
<p>Raman spectra of 6 nm (A) and 25 nm (B) commercial (black line) and acid-treated NDs (red line). FTIR spectra of 6 nm (C) and 25 nm (D) commercial (black line) and acid-treated NDs (red line). (E) Transmission electron micrographs from 6 nm and 25 nm acid-treated NDs. The diffraction patterns of the NDs are depicted in the inset of each image. Scale bar for 6 nm acid-treated NDs = 5 nm and scale bar for 25 nm acid-treated NDs = 20 nm.</p
Acid-treated 6 nm NDs are internalized in T24 cell by 2 hours.
<p>T24 cells were treated different concentrations of 6 nm acid-treated FITC labeled of unlabeled NDs for 1 hour. The cells were washed and analyzed by flow cytometry. The gated cells were evaluated for FITC fluorescence and the overlayed histograms (A) are depicted. The experiment was performed at least three independent times in duplicates. The result of one representative experiment is depicted. (B) The mean fluorescence intensity (MFI) for samples treated with FITC-labeled NDs was evaluated. The MFI of the untreated sample (μg/mL) was considered 1 and the MFI for all other samples were normalized to the untreated sample and plotted as mean ± SEM. Data are representative of at least three independent experiments. *<i>P<0</i>.<i>01</i>, **<i>P<0</i>.<i>0001</i> was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Scatter plots of Side scatter (SSC) vs FITC fluorescence of gated cells are depicted. The experiment was performed at least three independent times in duplicates. The result of one representative experiment is depicted. (D-G) T24 bladder cells were treated with different concentrations of FITC labeled 6 nm NDs for 1 hour (D-E) or with 100 μg/mL for different periods of time (F). The cells were fixed and permeabilized. The nuclei of the cells were stained with DAPI and actin filaments were stained with phalloidin conjugated to Alexa fluor 660. The cells were then observed by confocal microscopy. The experiment was performed at least three independent times and the result of one representative experiment is depicted. To determine the internalization of NDs in T24 bladder cells (E), z-stack analysis was performed and a few representative stacks of one image are shown. For the kinetic analysis (F), T24 bladder cells were treated with 100 μg/mLof FITC-labeled 6nm NDs for different periods of time. Z-stack analysis was performed and one representative image from the middle of the stack is depicted below. NDs associated with the cell membrane (*) or internalized in the cells (◆) are shown. Scale bar = 25 μm. The % of cells with NDs internalized was plotted (G) as mean ± SEM.</p
Acid-treated 6 nm NDs can kill extracellular and intracellular <i>E</i>. <i>coli</i>.
<p>(A) <i>E</i>. <i>coli</i> was treated with different concentrations of acid treated 6 nm or 25 nm NDs. Treatment with amoxicillin was used as a positive control. Following incubations, the samples were plated on sterile Luria agar and the colony forming units (CFU)/mL were enumerated. The CFU/mL for the 0 μg/mL sample was determined 100%. The CFU/mL counts for ND treated samples were determined relative to the 0 μg/mL sample. Data is represented as mean ± SEM. *<i>P<0</i>.<i>001</i>, **<i>P<0</i>.<i>0001</i> vs 0 μg/mL was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. (B) T24 cells were treated with 6 nm or 25 nm acid-treated NDs for 4 hours and analyzed by TEM. NDs are indicated by arrows. Scale bar = 500 nm. (C) T24 cells were infected with <i>E</i>. <i>coli</i>, followed by treatment with gentamicin to kill extracellular bacteria. Following gentamicin treatment, the infected T24 cells were treated with 6 nm or 25 nm acid treated NDs for 24 hours. The cells were then lysed and intracellular <i>E</i>. <i>coli</i> were enumerated. The CFU/mL for the 0 μg/mL sample was determined 100%. The CFU/mL for ND treated samples were determined relative to the 0 μg/mL sample. Data are representative of at least three independent experiments and depicted as mean ± SEM. *<i>P<0</i>.<i>01</i>, **<i>P<0</i>.<i>0001</i> compared to 0 μg/mL of the corresponding acid-treated ND was determined by two-way ANOVA followed by Tukey’s multiple comparisons test.</p
Internalization of acid-treated 6 nm NDs are required for efficient killing of intracellular bacteria.
<p>(A) T24 cells were treated with different concentrations of cytochalasin D for 1 hour followed by treatment with 100 μg/mL of NDs for 2 hours. The cells were fixed, permeabilized and the nuclei of the cells were stained with DAPI. Actin was stained with phalloidin conjugated to Alexa fluor 660. The cells were then observed by confocal microscopy and subjected to z-stack analysis to determine internalization of NDs. NDs associated with the cell membrane (*) or internalized in the cells (◆) are shown. The experiment was performed at least three independent times and the result of one representative experiment is depicted. Scale bar = 25 μm. (B) T24 cells were infected with <i>E</i>. <i>coli</i>, followed by treatment with gentamicin to kill extracellular bacteria. After gentamicin treatment, the infected T24 cells were treated with different concentrations of cytochalasin D for 1 hour followed by treatment with 6 nm or 25 nm acid treated NDs for 24 hours. The cells were then lysed and intracellular <i>E</i>. <i>coli</i> were enumerated. The cfu counts for the untreated sample was determined 100%. The CFU counts for ND treated samples were normalized to the 0 μg/mL sample. Data are representative of at least three independent experiments and depicted as mean ± SEM. *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>0001</i> compared to 0 μg/mL of the corresponding acid-treated ND sample was determined by two-way ANOVA followed by Tukey’s multiple comparisons test.</p
Comparison of % of internalized bacteria in cells treated with 6 nm or 25 nm NDs.
<p>Comparison of % of internalized bacteria in cells treated with 6 nm or 25 nm NDs.</p
pH and zeta potential measurements of 6 nm and 25 nm commercial and acid-treated NDs.
<p>pH and zeta potential measurements of 6 nm and 25 nm commercial and acid-treated NDs.</p
Comparison of cytotoxicity of acid treated 6 nm and 25 nm NDs.
<p>Comparison of cytotoxicity of acid treated 6 nm and 25 nm NDs.</p