Graphical abstract
Highlights
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Mesoporous silica nanoparticle (MSN) loading in poly(methyl methacrylate) (PMMA) changed the mechanical properties depending on the incorporated concentration.
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An anti-adherent effect against Candida albicans and Streptococcus oralis was observed without cytotoxicity in MSN-incorporated PMMA.
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A long-term anti-microbial effect was observed over 2 weeks due to the slow release of amphotericin B after loading them into MSN-incorporated PMMA.
Abstract
Objective
Poly(methyl methacrylate) (PMMA) used as removable denture bases or orthodontic appliances has relatively poor antimicrobial properties, which accelerate oral infection and induce unfavorable odors. Mesoporous silica nanoparticles (MSNs) have been highlighted as a potential additive to overcome this issue because of their drug-loading capacity. Here, we present the long-term antimicrobial effect of MSN-incorporated PMMA with drug-loading capacity.
Methods
After the MSNs were characterized, MSN incorporation into chemically activated PMMA (0.5, 1, 2.5 or 5 wt%) relative to the methyl methacrylate powder by mass was fabricated into a rectangular specimen (1.4 × 3.0 × 19.0 mm) for a 3-point flexural test at a speed of 1 mm/min or a disk ( ∅ = 11.5 mm and d = 1.5 mm) for investigation of its antimicrobial effects.
Results
A typical spherical morphology with a well-ordered mesoporous structure of the MSNs was visualized and is beneficial for loading drugs and combining in matrixes. Among the tested levels of MSN incorporation in PMMA (0.5, 1, 2.5 or 5 wt%), only 5 wt% decreased the flexural strength (p < 0.05), whereas the flexural modulus was not significantly decreased (p > 0.05). The surface roughness and surface energy were increased with 2.5 wt% or 5 wt% incorporation. An anti-adherent effect against Candida albicans and Streptococcus oralis after 1 h of attachment was only observed with 2.5 and 5 wt% incorporation compared to a lack of MSNs (p < 0.05). A long-term antimicrobial effect was observed for 2 weeks with 2.5 wt% MSN-incorporated PMMA when amphotericin B was loaded into the MSNs on the PMMA surface.
Significance
The long-term antimicrobial performance after loading amphotericin B into the MSN-incorporated PMMA suggests the potential clinical usefulness of MSN-incorporated PMMA resin.
1
Introduction
Poly(methyl methacrylate) (PMMA), clinically proven over the last 70 years, has been used as removable denture base materials, orthodontic appliance, and provisional resin crowns due to its easy fabrication process, appropriate mechanical properties and relatively low price . However, PMMA suffers from poor antimicrobial properties, which could accelerate oral infection and induce an unfavorable odor . One of the most recent intriguing research contributions that overcomes this problem is the development of nano-additives, either in the form of nanoparticles, nanofibers, or nanotubes .
Silver nanoparticles were incorporated into PMMA because of their antimicrobial effect, but they reduced the mechanical properties of the material or did not exert continuous antimicrobial effects after releasing silver ions . Quaternary ammonium poly(ethylenimine) nanoparticles were introduced into PMMA but lacked a long-term antimicrobial effect . Graphene oxide and carbon nanotubes reportedly possess antimicrobial activity and could therefore be used as antimicrobial nano-additives in PMMA . However, the black color from graphene oxide would limit the use of this material in esthetic dental materials. A photocatalytic bactericidal effect was observed when a nano-titanium dioxide and nano-silicon dioxide mixture was added to PMMA, but the material required an ultraviolet light source, which could degrade PMMA with prolonged exposure .
Mesoporous silica nanoparticles (MSNs) have been highlighted as potential nano-additives because of their biocompatibility, as well as their loading (charging) and delivery capacity for biomolecules due to their high surface area, high total pore volume and nanoscale morphological features, which are beneficial for anchorage in the PMMA matrix . The loading (charging) capacity of a drug carrier is an important aspect for maintaining a continuous antimicrobial effect in the oral cavity, which is an essential asset for clinical availability .
Amphotericin B is an effective antimicrobial drug that is taken intraorally as a rendered dental (bio)-material to prohibit fungal growth or infections; its FDA-approved biosafety and slow release profile result from its low solubility in aqueous systems, including bodily fluids . Therefore, current research is focused on incorporating this drug into nanocarriers or biodegradable polymers for a long-term antimicrobial dental/bio-material .
Here, we focus on the incorporation of MSNs into PMMA to induce antimicrobial effects. First, we fabricated MSNs and incorporated them into PMMA at loadings of up to 5 weight (wt)% and evaluated the anti-adherent effects against Candida albicans and Streptococcus oralis , as well as the mechanical properties using 3-point flexural and hardness tests. Furthermore, to induce an antifungal effect upon culturing with C. albicans , Amphotericin B was loaded into the specimens, and the antifungal effects were observed in specimens aged for up to 28 days. The cytotoxicity was investigated using human oral keratinocytes according to ISO 7405 to evaluate the adverse effects on oral mucosa.
The aim of this study was to evaluate the mechanical properties, antimicrobial activity, and cytotoxicity of MSN-incorporated PMMA with or without loading amphotericin B. Our null hypothesis states that the mechanical properties and antimicrobial effects observed in the MSN-incorporated PMMA do not differ significantly from those observed in PMMA without MSNs. Our second null hypothesis states that the amphotericin B-loaded ‘MSN-incorporated PMMA’ cannot significantly induce antifungal effects against C. albicans .
2
Materials and methods
2.1
Fabrication of MSNs
MSNs were prepared by a modified Stöber method. Briefly, 0.2 g of CTAB was dissolved in 96 mL of dH 2 O containing 0.7 mL of 2 M NaOH . The solution was heated to 80 °C, followed by the fast addition of 1.5 mL of TEOS, and the solution was then stirred for 2 h. The precipitated MSNs were collected by centrifugation at 8000 rpm, washed 3 times with water/ethanol solutions and then dried at 70 °C overnight. Finally, the dried MSNs were heat-treated at 550 °C for 5 h under an air flow. The particle size and morphology of the samples were investigated by transmission electron microscopy (TEM; 7100, JEOL, Japan). The pore volume and pore size were analyzed from N 2 adsorption-desorption measurements at −196.15 °C on an automated analyzer (Quadrasorb SI, Quantachrome Instruments Ltd., Boynton Beach, FL, USA) on the basis of the non-local density functional theory (NLDFT) method. The specific surface area was calculated according to the Brunauer–Emmett–Teller (BET) method. The zeta potential (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) was investigated in dimethyl sulfoxide (DMSO, Sigma) at 25 °C with an applied field strength of 20 V cm −1 .
2.2
MSN incorporation into PMMA
Commercially available, chemically activated, orthodontic PMMA resin (Orthocryl resin, Dentaurum, Ispringen, Germany; lot numbers: 452986B for powder and 450128 for liquid) was selected due to easy fabrication and clinical availability. In addition, heat-activated PMMA was not selected to avoid possible thermal damage to the MSNs during heat polymerization. Furthermore, denture resin was also ruled out because it contains many polymer pigments, which are an uncontrollable barrier to tailoring the surface characteristics with MSN incorporation. The MSNs were incorporated at 0.5, 1.0, 2.5, and 5.0 wt% with respect to the PMMA powder. After the nanoparticles were dispersed in the MMA liquid using sonication, the PMMA powder was immediately mixed together in a powder (g)-to-liquid (mL) ratio of 1.2:1. After additional polymerization at 40 °C for 30 min (Multicure, Vertex, Zeist, Netherlands) under 2.2 bar, the bar specimens for the mechanical properties test were prepared by cutting the polymerized blocks (4 × 15 × 20 mm) and then polishing them with 2400 grit SiC paper to the final dimensions (1.4 × 3.0 × 19 mm). Disk specimens ( ∅ = 11.5 mm and d = 1.5 mm) were prepared and polished with 800 grit SiC paper for other investigations. For the microbial and in vitro cytotoxicity tests, specimens were sterilized with ethylene oxide (EO) gas.
2.3
Mechanical properties
The prepared bar specimens were positioned on a jig with a span length of 14 mm in a universal testing machine (Instron 5966, MA, USA) with a 500-N load cell. The flexural strength and elastic modulus were determined at a crosshead speed of 1.0 mm/min (n = 10) . The Vickers hardness (HM-221, Mitutoyo, Tokyo, Japan) with 300 gf (2.94 N) was measured in five different spots on each specimen and then averaged. Five specimens were used to determine the hardness (n = 5).
2.4
Characterization of the MSN-incorporated PMMA
The surface morphology and composition were characterized by scanning electron microscopy (Sigma 500, ZEISS, Jena, Germany) at 3 kV and energy-dispersive spectroscopy (EDS, UltraDry, Thermo Fisher, Waltham, MA, USA) at 15 kV after Pt plasma coating for 5 min by an ion sputter (IB-3 Eiko, Tokyo, Japan). The Si ions released from a disk specimen immersed in YM broth (Difco, BD, Franklin Lakes, NJ, USA) (2 mL) were analyzed at determined times (1, 3, and 24 h; 2, 4, 7, 14, and 28 days) by inductively coupled plasma atomic emission spectrometry (Optima 4300 DV, PerkinElmer, Waltham, MA, n = 3) after replacing the old media with fresh media. The surface energy (n = 5, Phoenix 300, SEO, Suwonsi, Gyunggido, Korea) was measured according to the Owens–Wendt method, the surface roughness (n = 5, Ra) was determined at a speed of 0.5 mm/s with 4.0 mm of scanning (SJ-400, Mitutoyo, Japan). The details of the measurement procedures are described elsewhere .
2.5
Antimicrobial effect
For the anti-adherent assay, C. albicans (ATCC 10231, USA) and S. oralis (ATCC 9811) were grown according to the manufacturer’s protocol, and 100 μL of a 5 × 10 7 /mL microbial culture in the log phase of growth was seeded onto the surface of a disk specimen (n = 3). After a 1 h period to allow for attachment at 37 °C, the disk was washed with PBS (Welgene, Gyeongsan, Gyeongsangbuk-do, Korea) three times to detach any non-adherent microbes. After 12 or 3 h of incubation in 2 mL of the proper culture media in 12 wells for C. albicans and S. oralis , 200 μL of PrestoBlue (Molecular probes, USA) was added to each well and further incubated for 1.5 h. The liquid (100 μL) was transferred to a 96-well plate. To observe the antimicrobial effect of the specimens when co-culturing with microbial species, 2 mL of a 10 6 /mL microbial culture in the log phase of growth with the specimens were incubated for 5 h after PrestoBlue was added at 10%. The absorbance was read at 570 nm using a microplate reader (BioTek, Winooski, VT, USA) and normalized to the values at 600 nm for each well to enumerate the attached or alive microbes (n = 3). To confirm the correlation between the normalized absorbance in the PrestoBlue assay and the microbial numbers, the colony-forming units (CFUs) were serially counted, and the details of this method are described elsewhere . The assays were independently performed in triplicate. The representative means and SDs were recorded.
2.6
Loading and releasing of amphotericin B
Disk specimens were incubated at room temperature in 2 mL of amphotericin B (Sigma, St. Louis, MO, USA) in DMSO (Sigma) (0.625 mg/mL) for determined time periods (n = 3, 15 min, 30 min, 1 h, 3 h, 4 h, and 6 h), and the remaining drug solution was collected to determine the amount loaded in each specimen. The loaded amount was measured by the absorbance at 416 nm using a microplate (BioTek). Standard curves were generated from prepared samples with known concentrations of amphotericin B in DMSO. To determine the released amount, the disk specimen in which the drug was loaded for 6 h was immersed in 2 mL of YM broth (Difco) in a humidified incubator at 37 °C with 5% CO 2 . Subsequently, 2 mL of the elution solution (n = 3) was collected at determined time points (0, 2, 4, and 8 h; 1, 3, 7, 14, 28, 42, and 56 d) after replacing with fresh solution; the collected solutions were subsequently stored at −80 °C until further analysis. Amphotericin B was solubilized by mixing the eluate samples with DMSO at 10:90 to disperse the micelles. The released amount was measured based on the absorbance at 416 nm using a microplate (BioTek). Standard curves were generated from prepared samples with known concentrations of amphotericin B in a solution of YM broth-DMSO (1:9). The visual spectroscopy system had a sensitivity of 1 μg/mL (1 ppm) for amphotericin B in both the loading and releasing tests. The assays were independently performed in triplicate. The representative means and SDs were recorded.
2.7
Long-term antimicrobial effect after loading
Amphotericin B-loaded specimens (0 wt% and 2.5 wt% MSN) were aged in a humidified incubator at 37 °C with 5% CO 2 for up to 28 days in YM broth. After aging, the specimens were gathered at the determined time points described for the release test, washed with PBS (Welgene) three times, and dried in a vacuum desiccator; EO gas sterilization was performed. The antimicrobial test under co-culturing conditions with C. albicans described above was performed after 5 h of culturing with 2 mL of 10 6 /mL (n = 3). Only C. albicans , 2.5 wt% MSN-incorporated PMMA and amphotericin B-loaded PMMA without MSNs were used to compare the results. The representative means and SDs were recorded after independent triplicate experiments.
2.8
Cytotoxicity test
Immortalized human oral keratinocytes (IHOKs) were kindly provided by Prof. Eun-Cheol Kim . After seeding the cells (n = 6, 1 × 10 4 cells/96-well plate) and incubating the plates at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air, the specimen extracts were added at 50% into 2X supplemented media (DMEM/F-12 (3:1)) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL). Extraction was performed in supplemented DMEM/F-12 media at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air according to ISO 10093-12 (3 cm 2 /mL). After 24 h of incubation, the WST assay based on the absorbance at 450 nm was performed according to previous methods . The culture conditions of the control consisted of 50% distilled water and the 2X supplemented media mentioned above. The representative means and SDs were recorded after independent triplicate experiments.
2.9
Statistical analysis
The data are expressed as the mean ± S.D. of at least three independent experiments. Statistical significance was evaluated by one-way analysis of variance with Tukey’s post hoc test using SPSS (Version 21.0; SPSS, Chicago, IL). A value of p < 0.05 was considered statistically significant.
2
Materials and methods
2.1
Fabrication of MSNs
MSNs were prepared by a modified Stöber method. Briefly, 0.2 g of CTAB was dissolved in 96 mL of dH 2 O containing 0.7 mL of 2 M NaOH . The solution was heated to 80 °C, followed by the fast addition of 1.5 mL of TEOS, and the solution was then stirred for 2 h. The precipitated MSNs were collected by centrifugation at 8000 rpm, washed 3 times with water/ethanol solutions and then dried at 70 °C overnight. Finally, the dried MSNs were heat-treated at 550 °C for 5 h under an air flow. The particle size and morphology of the samples were investigated by transmission electron microscopy (TEM; 7100, JEOL, Japan). The pore volume and pore size were analyzed from N 2 adsorption-desorption measurements at −196.15 °C on an automated analyzer (Quadrasorb SI, Quantachrome Instruments Ltd., Boynton Beach, FL, USA) on the basis of the non-local density functional theory (NLDFT) method. The specific surface area was calculated according to the Brunauer–Emmett–Teller (BET) method. The zeta potential (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) was investigated in dimethyl sulfoxide (DMSO, Sigma) at 25 °C with an applied field strength of 20 V cm −1 .
2.2
MSN incorporation into PMMA
Commercially available, chemically activated, orthodontic PMMA resin (Orthocryl resin, Dentaurum, Ispringen, Germany; lot numbers: 452986B for powder and 450128 for liquid) was selected due to easy fabrication and clinical availability. In addition, heat-activated PMMA was not selected to avoid possible thermal damage to the MSNs during heat polymerization. Furthermore, denture resin was also ruled out because it contains many polymer pigments, which are an uncontrollable barrier to tailoring the surface characteristics with MSN incorporation. The MSNs were incorporated at 0.5, 1.0, 2.5, and 5.0 wt% with respect to the PMMA powder. After the nanoparticles were dispersed in the MMA liquid using sonication, the PMMA powder was immediately mixed together in a powder (g)-to-liquid (mL) ratio of 1.2:1. After additional polymerization at 40 °C for 30 min (Multicure, Vertex, Zeist, Netherlands) under 2.2 bar, the bar specimens for the mechanical properties test were prepared by cutting the polymerized blocks (4 × 15 × 20 mm) and then polishing them with 2400 grit SiC paper to the final dimensions (1.4 × 3.0 × 19 mm). Disk specimens ( ∅ = 11.5 mm and d = 1.5 mm) were prepared and polished with 800 grit SiC paper for other investigations. For the microbial and in vitro cytotoxicity tests, specimens were sterilized with ethylene oxide (EO) gas.
2.3
Mechanical properties
The prepared bar specimens were positioned on a jig with a span length of 14 mm in a universal testing machine (Instron 5966, MA, USA) with a 500-N load cell. The flexural strength and elastic modulus were determined at a crosshead speed of 1.0 mm/min (n = 10) . The Vickers hardness (HM-221, Mitutoyo, Tokyo, Japan) with 300 gf (2.94 N) was measured in five different spots on each specimen and then averaged. Five specimens were used to determine the hardness (n = 5).
2.4
Characterization of the MSN-incorporated PMMA
The surface morphology and composition were characterized by scanning electron microscopy (Sigma 500, ZEISS, Jena, Germany) at 3 kV and energy-dispersive spectroscopy (EDS, UltraDry, Thermo Fisher, Waltham, MA, USA) at 15 kV after Pt plasma coating for 5 min by an ion sputter (IB-3 Eiko, Tokyo, Japan). The Si ions released from a disk specimen immersed in YM broth (Difco, BD, Franklin Lakes, NJ, USA) (2 mL) were analyzed at determined times (1, 3, and 24 h; 2, 4, 7, 14, and 28 days) by inductively coupled plasma atomic emission spectrometry (Optima 4300 DV, PerkinElmer, Waltham, MA, n = 3) after replacing the old media with fresh media. The surface energy (n = 5, Phoenix 300, SEO, Suwonsi, Gyunggido, Korea) was measured according to the Owens–Wendt method, the surface roughness (n = 5, Ra) was determined at a speed of 0.5 mm/s with 4.0 mm of scanning (SJ-400, Mitutoyo, Japan). The details of the measurement procedures are described elsewhere .
2.5
Antimicrobial effect
For the anti-adherent assay, C. albicans (ATCC 10231, USA) and S. oralis (ATCC 9811) were grown according to the manufacturer’s protocol, and 100 μL of a 5 × 10 7 /mL microbial culture in the log phase of growth was seeded onto the surface of a disk specimen (n = 3). After a 1 h period to allow for attachment at 37 °C, the disk was washed with PBS (Welgene, Gyeongsan, Gyeongsangbuk-do, Korea) three times to detach any non-adherent microbes. After 12 or 3 h of incubation in 2 mL of the proper culture media in 12 wells for C. albicans and S. oralis , 200 μL of PrestoBlue (Molecular probes, USA) was added to each well and further incubated for 1.5 h. The liquid (100 μL) was transferred to a 96-well plate. To observe the antimicrobial effect of the specimens when co-culturing with microbial species, 2 mL of a 10 6 /mL microbial culture in the log phase of growth with the specimens were incubated for 5 h after PrestoBlue was added at 10%. The absorbance was read at 570 nm using a microplate reader (BioTek, Winooski, VT, USA) and normalized to the values at 600 nm for each well to enumerate the attached or alive microbes (n = 3). To confirm the correlation between the normalized absorbance in the PrestoBlue assay and the microbial numbers, the colony-forming units (CFUs) were serially counted, and the details of this method are described elsewhere . The assays were independently performed in triplicate. The representative means and SDs were recorded.
2.6
Loading and releasing of amphotericin B
Disk specimens were incubated at room temperature in 2 mL of amphotericin B (Sigma, St. Louis, MO, USA) in DMSO (Sigma) (0.625 mg/mL) for determined time periods (n = 3, 15 min, 30 min, 1 h, 3 h, 4 h, and 6 h), and the remaining drug solution was collected to determine the amount loaded in each specimen. The loaded amount was measured by the absorbance at 416 nm using a microplate (BioTek). Standard curves were generated from prepared samples with known concentrations of amphotericin B in DMSO. To determine the released amount, the disk specimen in which the drug was loaded for 6 h was immersed in 2 mL of YM broth (Difco) in a humidified incubator at 37 °C with 5% CO 2 . Subsequently, 2 mL of the elution solution (n = 3) was collected at determined time points (0, 2, 4, and 8 h; 1, 3, 7, 14, 28, 42, and 56 d) after replacing with fresh solution; the collected solutions were subsequently stored at −80 °C until further analysis. Amphotericin B was solubilized by mixing the eluate samples with DMSO at 10:90 to disperse the micelles. The released amount was measured based on the absorbance at 416 nm using a microplate (BioTek). Standard curves were generated from prepared samples with known concentrations of amphotericin B in a solution of YM broth-DMSO (1:9). The visual spectroscopy system had a sensitivity of 1 μg/mL (1 ppm) for amphotericin B in both the loading and releasing tests. The assays were independently performed in triplicate. The representative means and SDs were recorded.
2.7
Long-term antimicrobial effect after loading
Amphotericin B-loaded specimens (0 wt% and 2.5 wt% MSN) were aged in a humidified incubator at 37 °C with 5% CO 2 for up to 28 days in YM broth. After aging, the specimens were gathered at the determined time points described for the release test, washed with PBS (Welgene) three times, and dried in a vacuum desiccator; EO gas sterilization was performed. The antimicrobial test under co-culturing conditions with C. albicans described above was performed after 5 h of culturing with 2 mL of 10 6 /mL (n = 3). Only C. albicans , 2.5 wt% MSN-incorporated PMMA and amphotericin B-loaded PMMA without MSNs were used to compare the results. The representative means and SDs were recorded after independent triplicate experiments.
2.8
Cytotoxicity test
Immortalized human oral keratinocytes (IHOKs) were kindly provided by Prof. Eun-Cheol Kim . After seeding the cells (n = 6, 1 × 10 4 cells/96-well plate) and incubating the plates at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air, the specimen extracts were added at 50% into 2X supplemented media (DMEM/F-12 (3:1)) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL). Extraction was performed in supplemented DMEM/F-12 media at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air according to ISO 10093-12 (3 cm 2 /mL). After 24 h of incubation, the WST assay based on the absorbance at 450 nm was performed according to previous methods . The culture conditions of the control consisted of 50% distilled water and the 2X supplemented media mentioned above. The representative means and SDs were recorded after independent triplicate experiments.
2.9
Statistical analysis
The data are expressed as the mean ± S.D. of at least three independent experiments. Statistical significance was evaluated by one-way analysis of variance with Tukey’s post hoc test using SPSS (Version 21.0; SPSS, Chicago, IL). A value of p < 0.05 was considered statistically significant.
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