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International Journal of Nanomedicine Volume 15, 2020 - Issue
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Original Research

The Antibiofilm Activity and Mechanism of Nanosilver- and Nanozinc-Incorporated Mesoporous Calcium-Silicate Nanoparticles

Diya Leng1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
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Yan Li3 State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing, People’s Republic of ChinaORCID Iconhttps://orcid.org/0000-0002-0184-8439
ORCID Icon,
Jie Zhu4 Department of Stomatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
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Ruizhen Liang1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;5 Department of the Seventh Clinic, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
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Cuifeng Zhang1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
,
Yang Zhou1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
,
Mingming Li1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
,
Ying Wang1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
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Di Rong1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of China
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Daming Wu1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;2 Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of ChinaCorrespondence[email protected]
&
Jin Li1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, People’s Republic of China;6 Department of Oral Special Consultation, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, People’s Republic of ChinaCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-5891-8135
ORCID Icon show all
Pages 3921-3936 | Published online: 03 Jun 2020

Figures & data

Table 1 The Molar Ratio of Ag/Zn and the Weight of Silver Nitrate and Zinc Nitrate Added in the Synthesis of the Nanoparticles

Table 2 Surface Area (SBET), Pore Volume (VP), and Mean Pore Size (DP) of the Nanoparticles

Figure 1 Physicochemical properties of the prepared nanoparticles.

Notes: FE-SEM images of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (A and B); TEM images of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (C and D); EDS of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (E and F); nitrogen adsorption-desorption isotherm test and pore size distribution of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (G and H); ions release of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (I); pH measurement of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (J).

Abbreviations: Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated mesoporous calcium-silicate nanoparticles; FE-SEM, field emission scanning electron microscopy; TEM, transmission electron microscopy; EDS, energy dispersive spectrometry.

Figure 1 Physicochemical properties of the prepared nanoparticles.Notes: FE-SEM images of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (A and B); TEM images of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (C and D); EDS of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (E and F); nitrogen adsorption-desorption isotherm test and pore size distribution of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (G and H); ions release of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (I); pH measurement of Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs (J).Abbreviations: Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated mesoporous calcium-silicate nanoparticles; FE-SEM, field emission scanning electron microscopy; TEM, transmission electron microscopy; EDS, energy dispersive spectrometry.

Figure 2 Representative TEM images of endocytosis of nanoparticles by E. faecalis.

Notes: (A) E. faecalis (×30,000); (B–F) E. faecalis cocultured with Ag/Zn(1:1)-MCSNs. ×30,000 (B); ×100,000 (C and E); ×200,000 (D and F).

Abbreviations: TEM, transmission electron microscopy; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated mesoporous calcium-silicate nanoparticles.

Figure 2 Representative TEM images of endocytosis of nanoparticles by E. faecalis.Notes: (A) E. faecalis (×30,000); (B–F) E. faecalis cocultured with Ag/Zn(1:1)-MCSNs. ×30,000 (B); ×100,000 (C and E); ×200,000 (D and F).Abbreviations: TEM, transmission electron microscopy; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated mesoporous calcium-silicate nanoparticles.

Figure 3 Antibacterial effects of the materials against planktonic E. faecalis.

Notes: Comparisons of CFUs count among the groups, including Negative, CH, MCSNs, Ag-MCSNs, Ag/Zn(9:1)-MCSNs, Ag/Zn(1:1)-MCSNs, Ag/Zn(1:9)-MCSNs and Zn/MCSNs. *P<0.05 when compared with the negative control group. **P<0.05 when compared with the MCSNs group.

Abbreviations: CFUs, colony forming units; CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 3 Antibacterial effects of the materials against planktonic E. faecalis.Notes: Comparisons of CFUs count among the groups, including Negative, CH, MCSNs, Ag-MCSNs, Ag/Zn(9:1)-MCSNs, Ag/Zn(1:1)-MCSNs, Ag/Zn(1:9)-MCSNs and Zn/MCSNs. *P<0.05 when compared with the negative control group. **P<0.05 when compared with the MCSNs group.Abbreviations: CFUs, colony forming units; CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 4 Representative colonial pictures of the materials against planktonic E.faecalis.

Notes: (A) negative control group; (B) CH group; (C) MCSNs group; (D) Ag-MCSNs group; (E) Ag/Zn(9:1)-MCSNs group; (F) Ag/Zn(1:1)-MCSNs group; (G) Ag/Zn(1:9)-MCSNs group; (H) Zn-MCSNs group.

Abbreviations: CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 4 Representative colonial pictures of the materials against planktonic E.faecalis.Notes: (A) negative control group; (B) CH group; (C) MCSNs group; (D) Ag-MCSNs group; (E) Ag/Zn(9:1)-MCSNs group; (F) Ag/Zn(1:1)-MCSNs group; (G) Ag/Zn(1:9)-MCSNs group; (H) Zn-MCSNs group.Abbreviations: CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 5 Continued.

Figure 5 Continued.

Figure 5 Representative E. faecalis biofilms (4-week old) on dentin surface after medication were scanned by SEM or FE-SEM (capital letters: ×3000 magnification; small letters: ×10,000 magnification).

Notes: (A and a) Negative control group; (B and b) CH group; (C and c) MTA group; (D and d) MCSNs group; (E and e) Ag-MCSNs group; (F and f) Zn-MCSNs group; (G and g) Ag/Zn(1:9)-MCSNs group; (H and h) Ag/Zn(1:1)-MCSNs group; (I and i) Ag/Zn(9:1)-MCSNs group.

Abbreviations: SEM, scanning electron microscopy; FE-SEM, field emission scanning electron microscopy; CH, calcium hydroxide; MTA, mineral trioxide aggregate; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 5 Representative E. faecalis biofilms (4-week old) on dentin surface after medication were scanned by SEM or FE-SEM (capital letters: ×3000 magnification; small letters: ×10,000 magnification).Notes: (A and a) Negative control group; (B and b) CH group; (C and c) MTA group; (D and d) MCSNs group; (E and e) Ag-MCSNs group; (F and f) Zn-MCSNs group; (G and g) Ag/Zn(1:9)-MCSNs group; (H and h) Ag/Zn(1:1)-MCSNs group; (I and i) Ag/Zn(9:1)-MCSNs group.Abbreviations: SEM, scanning electron microscopy; FE-SEM, field emission scanning electron microscopy; CH, calcium hydroxide; MTA, mineral trioxide aggregate; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 6 CLSM 3-dimensional reconstructions of E. faecalis biofilms (4-week old) after medication (green: live cells; red: dead cells).

Notes: (A) Negative control group; (B) CH group; (C) MTA group; (D) MCSNs group; (E) Ag-MCSNs group; (F) Zn-MCSNs group; (G) Ag/Zn(1:9)-MCSNs group; (H) Ag/Zn(1:1)-MCSNs group; (I) Ag/Zn(9:1)-MCSNs group.

Abbreviations: CLSM, confocal laser scanning microscope; CH, calcium hydroxide; MTA, mineral trioxide aggregate; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 6 CLSM 3-dimensional reconstructions of E. faecalis biofilms (4-week old) after medication (green: live cells; red: dead cells).Notes: (A) Negative control group; (B) CH group; (C) MTA group; (D) MCSNs group; (E) Ag-MCSNs group; (F) Zn-MCSNs group; (G) Ag/Zn(1:9)-MCSNs group; (H) Ag/Zn(1:1)-MCSNs group; (I) Ag/Zn(9:1)-MCSNs group.Abbreviations: CLSM, confocal laser scanning microscope; CH, calcium hydroxide; MTA, mineral trioxide aggregate; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 7 Representative FE-SEM images showing colonization of E. faecalis on the pretreated root canal walls (capital letters: ×3000 magnification; small letters: ×10,000 magnification).

Notes: (A and a) Negative control group; (B and b) CH group; (C and c) MCSNs group; (D and d) Ag-MCSNs group; (E and e) Zn-MCSNs group; (F and f) Ag/Zn(1:9)-MCSNs group; (G and g) Ag/Zn(1:1)-MCSNs group; (H and h) Ag/Zn(9:1)-MCSNs group.

Abbreviations: FE-SEM, field emission scanning electron microscopy; CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 7 Representative FE-SEM images showing colonization of E. faecalis on the pretreated root canal walls (capital letters: ×3000 magnification; small letters: ×10,000 magnification).Notes: (A and a) Negative control group; (B and b) CH group; (C and c) MCSNs group; (D and d) Ag-MCSNs group; (E and e) Zn-MCSNs group; (F and f) Ag/Zn(1:9)-MCSNs group; (G and g) Ag/Zn(1:1)-MCSNs group; (H and h) Ag/Zn(9:1)-MCSNs group.Abbreviations: FE-SEM, field emission scanning electron microscopy; CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 8 E. faecalis adherence and colonization on the pretreated root canal walls observed by CLSM (green: live cells; red: dead cells).

Notes: (A) Blank control group (no background fluorescence produced by dentin); (B) Negative group; (C) CH group; (D) MCSNs group; (E) Ag-MCSNs group; (F) Zn-MCSNs group; (G) Ag/Zn(1:9)-MCSNs group; (H) Ag/Zn(1:1)-MCSNs group; (I) Ag/Zn(9:1)-MCSNs group.

Abbreviations: CLSM, confocal laser scanning microscope; CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 8 E. faecalis adherence and colonization on the pretreated root canal walls observed by CLSM (green: live cells; red: dead cells).Notes: (A) Blank control group (no background fluorescence produced by dentin); (B) Negative group; (C) CH group; (D) MCSNs group; (E) Ag-MCSNs group; (F) Zn-MCSNs group; (G) Ag/Zn(1:9)-MCSNs group; (H) Ag/Zn(1:1)-MCSNs group; (I) Ag/Zn(9:1)-MCSNs group.Abbreviations: CLSM, confocal laser scanning microscope; CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 9 CCK8 test on the MC3T3-E1 cells.

Notes: (A) Culture for 1 d; (B) culture for 3 d; (C) culture for 7 d. *P<0.05 when compared with the negative control (decrease); &P<0.05 when compared with the negative control (increase); #P<0.05 when compared with the CCK8 reagent only group.

Abbreviations: CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 9 CCK8 test on the MC3T3-E1 cells.Notes: (A) Culture for 1 d; (B) culture for 3 d; (C) culture for 7 d. *P<0.05 when compared with the negative control (decrease); &P<0.05 when compared with the negative control (increase); #P<0.05 when compared with the CCK8 reagent only group.Abbreviations: CH, calcium hydroxide; MCSNs, mesoporous calcium-silicate nanoparticles; Ag-MCSNs, nanosilver-incorporated MCSNs; Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated MCSNs; Zn-MCSNs, nanozinc-incorporated MCSNs.

Figure 10 Antimicrobial mechanism pattern diagram of Ag/Zn-MCSNs.

Abbreviation: Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated mesoporous calcium-silicate nanoparticles.

Figure 10 Antimicrobial mechanism pattern diagram of Ag/Zn-MCSNs.Abbreviation: Ag/Zn-MCSNs, nanosilver- and nanozinc-incorporated mesoporous calcium-silicate nanoparticles.

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