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Nanomedicine Volume 16, 2021 - Issue 30
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Research Article

Targeting Bacteria Causing Otitis Media Using Nanosystems Containing Nonspherical Gold Nanoparticles and Ceragenins

Suhanya V Prasad1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandView further author information
,
Ewelina Piktel1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandORCID Iconhttps://orcid.org/0000-0002-4531-7277View further author information
ORCID Icon,
Joanna Depciuch2 Institute of Nuclear Physics Polish Academy of Sciences, PL-31342, Krakow, PolandORCID Iconhttps://orcid.org/0000-0003-0168-1701View further author information
ORCID Icon,
Alexey Maximenko3 SOLARIS National Synchrotron Radiation Centre, Jagiellonian University, PL-30392, Krakow, PolandView further author information
,
Łukasz Suprewicz1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandView further author information
,
Tamara Daniluk1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandView further author information
,
Jakub Spałek4 Department of Pathology, Collegium Medicum, Jan Kochanowski University in Kielce, PL-25317, Kielce, Poland;5 Department of Otolaryngology, Head & Neck Surgery, Holy Cross Cancer Center in Kielce, PL-25734, Kielce, PolandView further author information
,
Urszula Wnorowska1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandView further author information
,
Piotr M Zielinski2 Institute of Nuclear Physics Polish Academy of Sciences, PL-31342, Krakow, PolandView further author information
,
Magdalena Parlinska-Wojtan2 Institute of Nuclear Physics Polish Academy of Sciences, PL-31342, Krakow, PolandView further author information
,
Paul B Savage6 Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT84604, USAView further author information
,
Sławomir Okła5 Department of Otolaryngology, Head & Neck Surgery, Holy Cross Cancer Center in Kielce, PL-25734, Kielce, PolandView further author information
,
Krzysztof Fiedoruk1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandView further author information
&
Robert Bucki1 Department of Medical Microbiology & Nanobiomedical Engineering, Medical University of Bialystok, PL-15222, Bialystok, PolandCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0001-7664-9226View further author information
ORCID Icon show all
Pages 2657-2678 | Received 07 Oct 2021, Accepted 08 Nov 2021, Published online: 26 Nov 2021

Figures & data

Figure 1. Physicochemical properties of developed nanosystems.

(A) Scheme of the AuP NPs biofunctionalization and ceragenin (CSA) immobilization process. (B) Overview scanning transmission electron microscopy image. (C) X-ray diffraction (XRD) taken for AuP NPs. Experimental (blue dots) and calculated (solid black line) Rietveld refinement plot of the XRD pattern for the AuP NPs. The red line (YOBS–YCALC) represents the difference between the observed and calculated data. Tick marks show allowed Bragg reflections. (D) FT-Raman spectra of nanosystems: AuP NPs + MHDA + CSA-13 (red spectrum); AuP NPs + MHDA + CSA-44 (blue spectrum); AuP NPs + MHDA + CSA-131 (green spectrum). (E) Ultraviolet-Vis spectra of AuP NPs (black spectrum) and AuP NPs + MHDA + CSA-13 (red spectrum); AuP NPs + MHDA + CSA-44 (blue spectrum); AuP NPs + MHDA + CSA-131 (green spectrum). (F) Thermogravimetric analysis and differential scanning calorimetry data of AuP NPs + MHDA and AuP@CSA-13 (red curve), AuP@CSA-44 (blue curve), AuP@CSA-131 (green curve), showing the solvent removal and decomposition of the product.

AuP NP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial; FT: Fourier transform; MHDA: Mercaptohexadecanoic acid.

Figure 1. Physicochemical properties of developed nanosystems. (A) Scheme of the AuP NPs biofunctionalization and ceragenin (CSA) immobilization process. (B) Overview scanning transmission electron microscopy image. (C) X-ray diffraction (XRD) taken for AuP NPs. Experimental (blue dots) and calculated (solid black line) Rietveld refinement plot of the XRD pattern for the AuP NPs. The red line (YOBS–YCALC) represents the difference between the observed and calculated data. Tick marks show allowed Bragg reflections. (D) FT-Raman spectra of nanosystems: AuP NPs + MHDA + CSA-13 (red spectrum); AuP NPs + MHDA + CSA-44 (blue spectrum); AuP NPs + MHDA + CSA-131 (green spectrum). (E) Ultraviolet-Vis spectra of AuP NPs (black spectrum) and AuP NPs + MHDA + CSA-13 (red spectrum); AuP NPs + MHDA + CSA-44 (blue spectrum); AuP NPs + MHDA + CSA-131 (green spectrum). (F) Thermogravimetric analysis and differential scanning calorimetry data of AuP NPs + MHDA and AuP@CSA-13 (red curve), AuP@CSA-44 (blue curve), AuP@CSA-131 (green curve), showing the solvent removal and decomposition of the product.AuP NP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial; FT: Fourier transform; MHDA: Mercaptohexadecanoic acid.
Figure 2. Bactericidal activity of tested compounds against otitis media-causing pathogens.

Activity of ceragenins CSA-13 (black squares), CSA-44 (red circles), CSA-131 (blue triangles) and ceragenin-containing nanosystems, AuP@CSA-13 (green squares), AuP@CSA-44 (purples circles) and AuP@CSA-131 (gold triangles) against (A)Haemophillus influenzae ATCC 49766, (B)Moraxella catarrhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 strains was evaluated. For each strain, logarithm of number of colonies outgrown [log (CFU)] (panels A1, B1 and C1) and concentration of compound reducing bacteria survival to 2 log (CFU/ml) was calculated (panels A2, B2 and C2). Results are presented as mean ± standard deviation from three replicates.

* and ∧ indicates statistical significance p < 0.05 when compared to untreated control and free ceragenins, respectively. (Where there is more than one *, the values overlap).

ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CFU: Colony-forming unit; CSA: Cationic steroid antimicrobial.

Figure 2. Bactericidal activity of tested compounds against otitis media-causing pathogens.Activity of ceragenins CSA-13 (black squares), CSA-44 (red circles), CSA-131 (blue triangles) and ceragenin-containing nanosystems, AuP@CSA-13 (green squares), AuP@CSA-44 (purples circles) and AuP@CSA-131 (gold triangles) against (A)Haemophillus influenzae ATCC 49766, (B)Moraxella catarrhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 strains was evaluated. For each strain, logarithm of number of colonies outgrown [log (CFU)] (panels A1, B1 and C1) and concentration of compound reducing bacteria survival to 2 log (CFU/ml) was calculated (panels A2, B2 and C2). Results are presented as mean ± standard deviation from three replicates.* and ∧ indicates statistical significance p < 0.05 when compared to untreated control and free ceragenins, respectively. (Where there is more than one *, the values overlap).ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CFU: Colony-forming unit; CSA: Cationic steroid antimicrobial.

Table 1. MIC and MBC of ceragenins CSA-13, CSA-44 and CSA-131 in free form and attached to the surface of AuPs against laboratory strains of H. influenzae, M. catarrhalis and S. pneumoniae.

Figure 3. Antibiofilm properties of ceragenins and ceragenin-containing nanosystems.

Prevention of biofilm formation by (A)Haemophillus influenzae ATCC 49766, (B)Moraxella cattarhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 bacteria treated with ceragenins CSA-13, CSA-44, CSA-131 and CSA-based nanosystems (AuP@CSA-13, AuP@CSA-44, AuP@CSA-131) at concentrations of 5 (gold bars), 20 (green stripped bars) and 50 μg/ml (blue bars) (panels A1, B1 and C1). On the basis of the recorded dose–response curves, mass of biofilm formed in the presence of 10 μg/ml of compound was calculated (panels A2, B2 and C2). Results are presented as mean ± standard deviation from three replicates.

* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively.

ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial.

Figure 3. Antibiofilm properties of ceragenins and ceragenin-containing nanosystems.Prevention of biofilm formation by (A)Haemophillus influenzae ATCC 49766, (B)Moraxella cattarhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 bacteria treated with ceragenins CSA-13, CSA-44, CSA-131 and CSA-based nanosystems (AuP@CSA-13, AuP@CSA-44, AuP@CSA-131) at concentrations of 5 (gold bars), 20 (green stripped bars) and 50 μg/ml (blue bars) (panels A1, B1 and C1). On the basis of the recorded dose–response curves, mass of biofilm formed in the presence of 10 μg/ml of compound was calculated (panels A2, B2 and C2). Results are presented as mean ± standard deviation from three replicates.* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively.ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial.
Figure 4. Bactericidal activity of tested compounds against otitis media-causing pathogens in the presence of 10% human cerumen buffer.

Activity of ceragenins CSA-13, CSA-44, CSA-131 and ceragenin-containing nanosystems AuP@CSA-13, AuP@CSA-44 and AuP@CSA-131 at doses of 0.5, 1 and 5 μg/ml was tested against (A)Haemophillus influenzae ATCC 49766, (B)Moraxella catarrhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619. Results are presented as mean ± standard deviation from three replicates.

* and ∧ indicate statistical significance p < 0.05 compared with untreated controls and free ceragenins, respectively.

#indicates statistical significance p < 0.05 compared with viability of bacteria in 10% CB and phosphate-buffered saline.

ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CB: Cerumen buffer; CSA: Cationic steroid antimicrobial; CFU: Colony-forming unit; PBS: Phosphate-buffered solution.

Figure 4. Bactericidal activity of tested compounds against otitis media-causing pathogens in the presence of 10% human cerumen buffer.Activity of ceragenins CSA-13, CSA-44, CSA-131 and ceragenin-containing nanosystems AuP@CSA-13, AuP@CSA-44 and AuP@CSA-131 at doses of 0.5, 1 and 5 μg/ml was tested against (A)Haemophillus influenzae ATCC 49766, (B)Moraxella catarrhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619. Results are presented as mean ± standard deviation from three replicates.* and ∧ indicate statistical significance p < 0.05 compared with untreated controls and free ceragenins, respectively. #indicates statistical significance p < 0.05 compared with viability of bacteria in 10% CB and phosphate-buffered saline.ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CB: Cerumen buffer; CSA: Cationic steroid antimicrobial; CFU: Colony-forming unit; PBS: Phosphate-buffered solution.
Figure 5. Generation of reactive oxygen species in otitis media-associated pathogens.

ROS-inducing abilities of ceragenins CSA-13, CSA-44, CSA-131 and ceragenin-containing nanosystems AuP@CSA-13, AuP@CSA-44 and AuP@CSA-44 at a dose of 5 μg/ml (gold bars), 20 μg/ml (green stripped bars) and 50 μg/ml (blue bars) were tested against (A)Haemophillus influenzae ATCC 49766, (B)Moraxella catarrhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 (panel C). Results are presented as mean ± standard error of the mean from three to six individual experiments with three replicates each.

* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively.

ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial; ROS: Reactive oxygen species.

Figure 5. Generation of reactive oxygen species in otitis media-associated pathogens.ROS-inducing abilities of ceragenins CSA-13, CSA-44, CSA-131 and ceragenin-containing nanosystems AuP@CSA-13, AuP@CSA-44 and AuP@CSA-44 at a dose of 5 μg/ml (gold bars), 20 μg/ml (green stripped bars) and 50 μg/ml (blue bars) were tested against (A)Haemophillus influenzae ATCC 49766, (B)Moraxella catarrhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 (panel C). Results are presented as mean ± standard error of the mean from three to six individual experiments with three replicates each.* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively.ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial; ROS: Reactive oxygen species.
Figure 6. Membrane-permeabilizing properties of ceragenins and cationic steroid antimicrobial-containing nanosystems.

Disruption of bacterial membrane in (A)Haemophillus influenzae ATCC 49766 and (B)Moraxella catarrhalis ATCC 25238 bacteria treated with ceragenins CSA-13, CSA-44, CSA-131 and CSA-based nanosystems (AuP@CSA-13, AuP@CSA-44 and AuP@CSA-131) at concentrations of 5 (gold bars), 20 (green stripped bars) and 50 μg/ml (blue bars). Results are presented as mean ± standard deviation from two individual experiments with three replicates each.

* and ∧ indicate statistical significance p < 0.05 compared with untreated control and free ceragenins, respectively.

ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial; NPN: N-phenyl-1-naphthylamine.

Figure 6. Membrane-permeabilizing properties of ceragenins and cationic steroid antimicrobial-containing nanosystems.Disruption of bacterial membrane in (A)Haemophillus influenzae ATCC 49766 and (B)Moraxella catarrhalis ATCC 25238 bacteria treated with ceragenins CSA-13, CSA-44, CSA-131 and CSA-based nanosystems (AuP@CSA-13, AuP@CSA-44 and AuP@CSA-131) at concentrations of 5 (gold bars), 20 (green stripped bars) and 50 μg/ml (blue bars). Results are presented as mean ± standard deviation from two individual experiments with three replicates each.* and ∧ indicate statistical significance p < 0.05 compared with untreated control and free ceragenins, respectively.ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial; NPN: N-phenyl-1-naphthylamine.
Figure 7. Release of intracellular content from cationic steroid antimicrobials and AuP@CSA-treated bacteria.

Release of proteins from (A)Haemophillus influenzae ATCC 49766, (B)Moraxella cattarhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 bacteria treated with ceragenins CSA-13, CSA-44, CSA-131 and CSA-based nanosystems (AuP@CSA-13, AuP@CSA-44, AuP@CSA-131) at concentrations of 5 (gold bars), 20 (green stripped bars) and 50 μg/ml (blue bars). Results are presented as mean ± standard deviation from three replicates.

* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively.

ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial.

Figure 7. Release of intracellular content from cationic steroid antimicrobials and AuP@CSA-treated bacteria.Release of proteins from (A)Haemophillus influenzae ATCC 49766, (B)Moraxella cattarhalis ATCC 25238 and (C)Streptococcus pneumoniae ATCC 49619 bacteria treated with ceragenins CSA-13, CSA-44, CSA-131 and CSA-based nanosystems (AuP@CSA-13, AuP@CSA-44, AuP@CSA-131) at concentrations of 5 (gold bars), 20 (green stripped bars) and 50 μg/ml (blue bars). Results are presented as mean ± standard deviation from three replicates.* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively.ATCC: American Type Culture Collection; AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial.
Figure 8. Decrease of Haemophillus influenzae-induced inflammation by ceragenins and ceragenin-containing nanosystems.

(A) Cytotoxicity of ceragenins CSA-13, CSA-44, CSA-131 and CSA-containing nanosystems AuP@CSA-13, AuP@CSA-44 and AuP@CSA-131 in relations to human keratinocytes (HaCaT) treated with 24 h with tested agents at concentrations ranging from 1 to 20 μg/ml. (B) Decrease of IL-8 release from human keratinocytes stimulated with H. influenzae ATCC 49766 bacteria and treated with tested agents for 24 h. Results are presented as mean ± standard deviation from three replicates.

* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively. (Where there is more than one *, the values overlap).

AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial.

Figure 8. Decrease of Haemophillus influenzae-induced inflammation by ceragenins and ceragenin-containing nanosystems. (A) Cytotoxicity of ceragenins CSA-13, CSA-44, CSA-131 and CSA-containing nanosystems AuP@CSA-13, AuP@CSA-44 and AuP@CSA-131 in relations to human keratinocytes (HaCaT) treated with 24 h with tested agents at concentrations ranging from 1 to 20 μg/ml. (B) Decrease of IL-8 release from human keratinocytes stimulated with H. influenzae ATCC 49766 bacteria and treated with tested agents for 24 h. Results are presented as mean ± standard deviation from three replicates.* and ∧ indicate statistical significance (p < 0.05) compared with untreated control and free ceragenins, respectively. (Where there is more than one *, the values overlap).AuP: Gold peanut-shaped nanoparticle; CSA: Cationic steroid antimicrobial.
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