Antifungal activity of dental resins containing amphotericin B-conjugated nanoparticles

Abstract

Objectives

To evaluate the antifungal activity, biocompatibility and mechanical properties of dental resins containing silica nanoparticles functionalized with amphotericin B (SNP-DexOxAmB) against five species of Candida.

Methods

Dental resin composites (Spectrum, Dentsply DeTrey, GmbH, Germany) having 2% (w/w) of SNP-DexOxAmB (SNPs of 5 and 80 nm, denoted as SNP5 and SNP80) were aged for 10, 20 and 30 days at 37 °C, in phosphate buffer saline buffer pH 7.4 (PBS). At different time, the antifungal activity was evaluated by a direct contact assay against 1 × 10 4 cells of Candida . The biocompatibility of the resins was tested against human fibroblasts, endothelial cells and red blood cells.

Results

Dental resins containing SNP5-DexOxAmB have high (1 × 10 4 cells killed in 5 h by ∼70 mg of dental resin composite containing 2% (w/w) of SNP-DexOxAmB) and durable (for at least 1 month) antifungal activity against five strains of Candida . The incorporation of the nanoparticles (NPs) had no significant change in the mechanical properties of the resin, specifically the flexural strength and modulus. Our results further show that the antifungal activity is mainly mediated by direct contact and not by leaching of NPs from the resin. Resins incorporating SNP5-DexOxAmB have longer-term antifungal activity than SNP80-DexOxAmB. The antimicrobial activity of resins with SNP5-DexOxAmB persists after 4 cycles of re-use and it is superior to the activity obtained for dental resins containing silver NPs. In addition, dental resins incorporating SNP5-DexOxAmB are non-cytotoxic against human skin fibroblasts and human umbilical vein endothelial cells, and non-hemolytic against human red blood cells.

Significance

The incorporation of SNP5-DexOxAmB in dental resins resulted in a non-cytotoxic composite with high and durable antifungal activity.

Introduction

Dental resin composites, which have been developed as an esthetic alternative to amalgam in early 60’s, are very popular in clinical dentistry as restorative materials. Typically, these materials are formed by both a polymeric matrix and inorganic particles ( e.g. silica) . Unfortunately, over time, these restorative materials tend to be colonized by microorganisms, such as bacteria and fungi . Fungal colonization is particularly significant in patients with HIV , diabetes and patients with tumors being treated with chemotherapy or radiation . For example, it has been reported that more than 80% of HIV-infected patients are susceptible to oral candidiasis . Candida albicans adheres to dental resin composites being the process influenced by several factors including surface roughness, surface hydrophobicity, composition of the material and by the composition of human saliva .

Several approaches have been described for the preparation of antimicrobial dental resin composites , including the ones based in the incorporation of silver-based materials and quaternary ammonium poly(ethylenimine) nanoparticles (NPs) . Unfortunately, little information is available concerning the potential antifungal properties of these approaches.

Amphotericin B (AmB) is a potent antifungal agent, approved by the FDA, widely used in clinical practice and effective against a large spectrum of fungi. Few resistant strains of fungi to AmB have been reported so far . Experimental data indicates that AmB associates with the ergosterol in the fungi membrane, forming pores and consequently disrupting the ionic gradient . Recently we reported a novel methodology to produce antifungal nanomaterials based on the permanent immobilization of AmB to silica NPs (SNPs) . SNPs were chosen due to their non-cytotoxicity, low price, high stability and durability and ease of modification by organosilane chemistry, allowing the incorporation of an array of different functional groups . We have demonstrated that these NPs have high antifungal activity against several species of Candida and can be reused for 5 cycles (each cycle of 8 h; exposure to 1 × 10 5 cells of C. albicans ) without losing their antifungal activity. The antifungal activity of the NPs was mediated by contact and not by the release of the AmB in the media. We hypothesize that the incorporation of these NPs in dental resins will render the composite material with antifungal properties. However, it is unclear the concentration needed to render the resin with antimicrobial activity. Furthermore, it is unknown the impact of the NP size in the antimicrobial activity of the resin (short- and long-term activity), leaching of the NPs, mechanical properties and cytotoxicity of the resin. The aim of the present study was to evaluate the mechanical, biocompatibility and antifungal activity of dental resin composites containing AmB-conjugated nanoparticles with different sizes.

Materials and methods

SNPs functionalized with AmB

SNPs with diameters of 5 and 80 nm were functionalized with AmB (SNP-AmB) according to a methodology previously described . Initially, SNP5 and SNP80 were silanized with amine-containing silane compounds (denoted as SNP80-NH 2 and SNP5-NH 2 ). AmB was immobilized covalently to oxidized dextran (DexOx) to improve AmB solubility in aqueous solutions and thus facilitate its immobilization onto SNPs. Then, DexOxAmB (12 mg/mL) with a degree of AmB incorporation of 15% was reacted with SNPs. The content of DexOxAmB conjugate immobilized on the surface of NPs was determined by the anthrone assay . The average diameter of SNPs suspended in 2 mL filtered milliQ water was determined by a dynamic light scattering method (DLS) using a Zeta Plus analyzer (Brookhaven), from six independent measurements. The zeta potential of SNPs was determined by a Zeta Plus analyzer, from three independent measurements. In this case, an aliquot (20 μL) of SNPs suspended in water (8 mg/mL) was added to 1.5 mL of 0.1 M MES buffer pH 5.5, and the zeta potential determined.

Incorporation of SNPs in dental resin composites

The samples were prepared by adding 2% (w/w) of SNPs (SNP5-DexOx, SNP5-DexOxAmB, SNP80-DexOx or SNP80-DexOxAmB) to the dental resin composite (Spectrum TPH3, Dentisply DeTrey, GmbH, Germany). The composition of the dental resin is summarized in Supplementary Table 1. After thoroughly mixing with a spatula, the samples (resin + NPs) (70 ± 5 mg in each well) were applied to the sidewalls of a 96 well plate (round bottom plate, Corning, Netherlands) vertically positioned and polymerized for 10 s using a SmartLitePS LED curing light (DENTSPLY DeTrey GmbH, Germany) at 460 nm with an average light output of 950 mW/cm 2 .

Evaluation of mechanical properties of the dental resin composites

The mechanical properties of the samples were evaluated according to a methodology reported elsewhere . Six samples from each experimental group were prepared using a glass mold (25 mm × 2 mm × 2 mm) and light-cured for 6 periods of 10 s along the horizontal length of the bulk on both sides of the mold. The photopolymerization was performed using a SmartLitePS LED curing light (see above). Samples were stored in PBS at 37 °C for 12 h before mechanical testing. Three point bending tests were carried out using a universal testing machine (Instron 5566) with a load cell of 10 kN and crosshead speed of 0.6 mm/min. Flexural strength and flexural modulus were calculated using Instron Bluehill software. The flexural strength was calculated according to σ = (3 Fl /2 bh 2 ). F is the maximal load (N) exerted on the specimen, l is the distance (mm) between the supports, b is the width (mm) of the specimen, h is the thickness (mm) of the specimen measured before testing. The deformation is determined by ɛ = (6 Yh / l 2 ) where Y is the maximum deflection of the center of the sample. The flexural or bending modulus was obtained from the slope of the stress/deformation curve. Statistical analyses were performed by one-way ANOVA followed by a Tukey’s test ( α = 0.05).

SEM analysis of dental resin composites

Resin composite with 2% (w/w) SNPs was pressed between two glass slides to obtain smooth surfaces. The disks were photo-polymerized for 10 s on both sides, using a SmartLitePS LED curing light. The disks were then sterilized by immersion in an ethanol solution (70%, v/v) for 2 min and dried. A 10 μL of Candida suspension (1 × 10 4 cells) was placed on the surface of the sample and dried under sterile conditions for 5 h at room temperature. The samples were then immersed in PBS to remove the cells loosely attached to the resin surface. The samples were fixed with glutaraldehyde 2.5% (30 min) and osmium tetroxide 1% (15 min) and dehydrated in ethanol solutions (30%, 50%, 70%, 90% and 100%, 10 min each) and in hexamethyldisilazane (10 min). An additional set of disks was processed as above and incubated for 48 h in 200 μL of Yeast Peptone Dextrose (YPD) medium. The samples were then fixed and processed as above. Finally, the samples were carbon coated by plasma vapor deposition and analyzed by a Hitachi SU-70 SEM, operated at 15 kV.

Aging of dental resin composites containing SNPs

The samples were aged in PBS during 10, 20 and 30 days at 37 °C. Each well of the microplate was filled with 250 μL of PBS which was replaced every 24 h. After this procedure the samples were dried under sterile conditions and tested as described previously.

Antimicrobial activity

SNPs in suspension

SNPs functionalized with DexOxAmB were tested in suspension against C. albicans (ATCC 10231), Candida tropicalis (ATCC 4563), Candida parapsilosis (ATCC 90018), Candida krusei ( Issatchenkia orientalis ) (ATCC 6258) and Candida glabrata (ATCC 90030). Candida species were incubated in YPD medium overnight, at 30 °C and mild agitation (150 rpm), before use. SNPs (from 50 μg/mL to 5 mg/mL) were incubated with 1 mL YPD containing 1 × 10 5 yeast cells for 8 h at 30 °C and 150 rpm orbital shaking. An aliquot of the medium was then serially diluted with sterile water and plated on YPD agar (1% yeast extract, 2% peptone, 2% dextrose, 2% agar). The plates were incubated at 30 °C for 24 h and the number of CFU was counted and compared with the control ( Candida incubated without SNPs). MICs were determined by the broth microdilution method according to the National Committee for Clinical Laboratory Standard (NCCLS) [M27A2E] guidelines.

Resin composites containing SNPs

The antifungal activity of dental composites containing SNPs was tested following a procedure previously reported . Briefly, resin samples having SNPs were prepared according to Section 2.4 in the sidewalls of a 96 well plate. The samples were then sterilized by immersion in an ethanol solution (70%, v/v) for 2 min and finally dried. Candida suspension (10 μL with 1 × 10 4 cells) was applied to the surface of the sample, after which the 96 well plate was positioned vertically and the samples were left to dry in a laminar flow cabinet at room temperature for 5 h to ensure direct contact between fungi and the surface. The plate was then positioned horizontally, 200 μL of YPD medium [supplemented with Penstrep (Lonza) solution at 0.5% (v/v)] added to each well and the plate placed in a microplate spectrophotometer (BioTek Synergy Mx, United States). The plate was incubated for 16 h at 30 °C (optimal temperature to grow C. albicans ) or 37 °C (to mimic body temperature) and the optical density (OD) at 600 nm measured every 30 min. After the incubation, an aliquot of the medium of each well was diluted and plated on YPD agar. The number of colony forming units (CFU) was counted after incubation of the plates at 30 °C or 37 °C for 24 h. The samples were then washed with ethanol to remove fragments of fungi and retested according to the previous methodology.

Cytotoxicity of composite resins containing SNP-DexOxAmB

Primary human skin fibroblasts were grown in DMEM supplemented with fetal bovine serum (FBS, 10% v/v), at 37 °C in a fully humidified air containing 5% CO 2 . The medium was changed every 2–3 days. Human umbilical vein endothelial cells (HUVECs, Lonza) were cultured in EGM-2 media (Lonza) being the medium changed every 2 days. Both cells were passaged after reaching 80% confluency. Cultures between the 3rd and 7th passages were used in the entire work.

The extraction assay was performed in resin samples prepared according to Section 2.4 in the sidewalls of a 96 well plate. Each well was filled with 250 μL of DMEM (supplemented with 10% FBS) or EGM-2 and the medium was collected after 24 and 48 h. The ratio between the surface area of the material and the volume of extraction medium was 3 cm 2 /mL. In parallel, human dermal fibroblasts or HUVECs were cultured in a 24 well plate for 24 h. The culture medium was then removed and replaced with the extract media (1 mL per well) for 24 h at 37 °C. Culture medium without extracts, incubated as described above, was used as a negative control. ATP was measured by a Celltiter-Glo Luminescent Cell Viability Assay (Promega).

For the contact assay, resin composites with SNPs were pressed between two glass slides to obtain smooth surfaces, photo-polymerized for 10 s on both sides, and immersed in an ethanol solution (70%, v/v) for 2 min and dried. The disks (diameter of 5 mm, approximately 30 mg) were kept in a PBS solution for 2 days and finally used as a culture substrate of HUVECs or fibroblasts for 24 h. The metabolic activity of the cells was determined at the end of 24 h by a MTT assay.

Hemocompatibility of composite resins

The hemocompatibility assay was preformed in resin samples prepared according to Section 2.4 in the sidewalls of a 96 well plate. Composite resins were immersed in 0.2 mL PBS for 12 h with orbital agitation (150 rpm) before use. The hemocompatibility of the composite resins was evaluated against human red blood cells (RBC). RBCs were isolated from human umbilical cord blood by centrifugation at room temperature, for 10 min at 600 × g , and then ressuspended in PBS 7.2 (2 × 10 8 cells/mL). Composite resins without SNPs were used as negative controls. PBS containing 20 mM SDS was used as a positive control. RBCs (2 × 10 8 cells/mL, 200 μL) were incubated with each composite resin for 24 h at 37 °C under orbital shaking (150 rpm). At the end, the cell suspension was collected and centrifuged for 10 min at 1000 × g and 4 °C. The free hemoglobin in the supernatant was quantified by the measurement of the absorbance at 540 nm.

Materials and methods

SNPs functionalized with AmB

SNPs with diameters of 5 and 80 nm were functionalized with AmB (SNP-AmB) according to a methodology previously described . Initially, SNP5 and SNP80 were silanized with amine-containing silane compounds (denoted as SNP80-NH 2 and SNP5-NH 2 ). AmB was immobilized covalently to oxidized dextran (DexOx) to improve AmB solubility in aqueous solutions and thus facilitate its immobilization onto SNPs. Then, DexOxAmB (12 mg/mL) with a degree of AmB incorporation of 15% was reacted with SNPs. The content of DexOxAmB conjugate immobilized on the surface of NPs was determined by the anthrone assay . The average diameter of SNPs suspended in 2 mL filtered milliQ water was determined by a dynamic light scattering method (DLS) using a Zeta Plus analyzer (Brookhaven), from six independent measurements. The zeta potential of SNPs was determined by a Zeta Plus analyzer, from three independent measurements. In this case, an aliquot (20 μL) of SNPs suspended in water (8 mg/mL) was added to 1.5 mL of 0.1 M MES buffer pH 5.5, and the zeta potential determined.

Incorporation of SNPs in dental resin composites

The samples were prepared by adding 2% (w/w) of SNPs (SNP5-DexOx, SNP5-DexOxAmB, SNP80-DexOx or SNP80-DexOxAmB) to the dental resin composite (Spectrum TPH3, Dentisply DeTrey, GmbH, Germany). The composition of the dental resin is summarized in Supplementary Table 1. After thoroughly mixing with a spatula, the samples (resin + NPs) (70 ± 5 mg in each well) were applied to the sidewalls of a 96 well plate (round bottom plate, Corning, Netherlands) vertically positioned and polymerized for 10 s using a SmartLitePS LED curing light (DENTSPLY DeTrey GmbH, Germany) at 460 nm with an average light output of 950 mW/cm 2 .

Evaluation of mechanical properties of the dental resin composites

The mechanical properties of the samples were evaluated according to a methodology reported elsewhere . Six samples from each experimental group were prepared using a glass mold (25 mm × 2 mm × 2 mm) and light-cured for 6 periods of 10 s along the horizontal length of the bulk on both sides of the mold. The photopolymerization was performed using a SmartLitePS LED curing light (see above). Samples were stored in PBS at 37 °C for 12 h before mechanical testing. Three point bending tests were carried out using a universal testing machine (Instron 5566) with a load cell of 10 kN and crosshead speed of 0.6 mm/min. Flexural strength and flexural modulus were calculated using Instron Bluehill software. The flexural strength was calculated according to σ = (3 Fl /2 bh 2 ). F is the maximal load (N) exerted on the specimen, l is the distance (mm) between the supports, b is the width (mm) of the specimen, h is the thickness (mm) of the specimen measured before testing. The deformation is determined by ɛ = (6 Yh / l 2 ) where Y is the maximum deflection of the center of the sample. The flexural or bending modulus was obtained from the slope of the stress/deformation curve. Statistical analyses were performed by one-way ANOVA followed by a Tukey’s test ( α = 0.05).

SEM analysis of dental resin composites

Resin composite with 2% (w/w) SNPs was pressed between two glass slides to obtain smooth surfaces. The disks were photo-polymerized for 10 s on both sides, using a SmartLitePS LED curing light. The disks were then sterilized by immersion in an ethanol solution (70%, v/v) for 2 min and dried. A 10 μL of Candida suspension (1 × 10 4 cells) was placed on the surface of the sample and dried under sterile conditions for 5 h at room temperature. The samples were then immersed in PBS to remove the cells loosely attached to the resin surface. The samples were fixed with glutaraldehyde 2.5% (30 min) and osmium tetroxide 1% (15 min) and dehydrated in ethanol solutions (30%, 50%, 70%, 90% and 100%, 10 min each) and in hexamethyldisilazane (10 min). An additional set of disks was processed as above and incubated for 48 h in 200 μL of Yeast Peptone Dextrose (YPD) medium. The samples were then fixed and processed as above. Finally, the samples were carbon coated by plasma vapor deposition and analyzed by a Hitachi SU-70 SEM, operated at 15 kV.

Aging of dental resin composites containing SNPs

The samples were aged in PBS during 10, 20 and 30 days at 37 °C. Each well of the microplate was filled with 250 μL of PBS which was replaced every 24 h. After this procedure the samples were dried under sterile conditions and tested as described previously.

Antimicrobial activity

SNPs in suspension

SNPs functionalized with DexOxAmB were tested in suspension against C. albicans (ATCC 10231), Candida tropicalis (ATCC 4563), Candida parapsilosis (ATCC 90018), Candida krusei ( Issatchenkia orientalis ) (ATCC 6258) and Candida glabrata (ATCC 90030). Candida species were incubated in YPD medium overnight, at 30 °C and mild agitation (150 rpm), before use. SNPs (from 50 μg/mL to 5 mg/mL) were incubated with 1 mL YPD containing 1 × 10 5 yeast cells for 8 h at 30 °C and 150 rpm orbital shaking. An aliquot of the medium was then serially diluted with sterile water and plated on YPD agar (1% yeast extract, 2% peptone, 2% dextrose, 2% agar). The plates were incubated at 30 °C for 24 h and the number of CFU was counted and compared with the control ( Candida incubated without SNPs). MICs were determined by the broth microdilution method according to the National Committee for Clinical Laboratory Standard (NCCLS) [M27A2E] guidelines.

Resin composites containing SNPs

The antifungal activity of dental composites containing SNPs was tested following a procedure previously reported . Briefly, resin samples having SNPs were prepared according to Section 2.4 in the sidewalls of a 96 well plate. The samples were then sterilized by immersion in an ethanol solution (70%, v/v) for 2 min and finally dried. Candida suspension (10 μL with 1 × 10 4 cells) was applied to the surface of the sample, after which the 96 well plate was positioned vertically and the samples were left to dry in a laminar flow cabinet at room temperature for 5 h to ensure direct contact between fungi and the surface. The plate was then positioned horizontally, 200 μL of YPD medium [supplemented with Penstrep (Lonza) solution at 0.5% (v/v)] added to each well and the plate placed in a microplate spectrophotometer (BioTek Synergy Mx, United States). The plate was incubated for 16 h at 30 °C (optimal temperature to grow C. albicans ) or 37 °C (to mimic body temperature) and the optical density (OD) at 600 nm measured every 30 min. After the incubation, an aliquot of the medium of each well was diluted and plated on YPD agar. The number of colony forming units (CFU) was counted after incubation of the plates at 30 °C or 37 °C for 24 h. The samples were then washed with ethanol to remove fragments of fungi and retested according to the previous methodology.

Cytotoxicity of composite resins containing SNP-DexOxAmB

Primary human skin fibroblasts were grown in DMEM supplemented with fetal bovine serum (FBS, 10% v/v), at 37 °C in a fully humidified air containing 5% CO 2 . The medium was changed every 2–3 days. Human umbilical vein endothelial cells (HUVECs, Lonza) were cultured in EGM-2 media (Lonza) being the medium changed every 2 days. Both cells were passaged after reaching 80% confluency. Cultures between the 3rd and 7th passages were used in the entire work.

The extraction assay was performed in resin samples prepared according to Section 2.4 in the sidewalls of a 96 well plate. Each well was filled with 250 μL of DMEM (supplemented with 10% FBS) or EGM-2 and the medium was collected after 24 and 48 h. The ratio between the surface area of the material and the volume of extraction medium was 3 cm 2 /mL. In parallel, human dermal fibroblasts or HUVECs were cultured in a 24 well plate for 24 h. The culture medium was then removed and replaced with the extract media (1 mL per well) for 24 h at 37 °C. Culture medium without extracts, incubated as described above, was used as a negative control. ATP was measured by a Celltiter-Glo Luminescent Cell Viability Assay (Promega).

For the contact assay, resin composites with SNPs were pressed between two glass slides to obtain smooth surfaces, photo-polymerized for 10 s on both sides, and immersed in an ethanol solution (70%, v/v) for 2 min and dried. The disks (diameter of 5 mm, approximately 30 mg) were kept in a PBS solution for 2 days and finally used as a culture substrate of HUVECs or fibroblasts for 24 h. The metabolic activity of the cells was determined at the end of 24 h by a MTT assay.

Hemocompatibility of composite resins

The hemocompatibility assay was preformed in resin samples prepared according to Section 2.4 in the sidewalls of a 96 well plate. Composite resins were immersed in 0.2 mL PBS for 12 h with orbital agitation (150 rpm) before use. The hemocompatibility of the composite resins was evaluated against human red blood cells (RBC). RBCs were isolated from human umbilical cord blood by centrifugation at room temperature, for 10 min at 600 × g , and then ressuspended in PBS 7.2 (2 × 10 8 cells/mL). Composite resins without SNPs were used as negative controls. PBS containing 20 mM SDS was used as a positive control. RBCs (2 × 10 8 cells/mL, 200 μL) were incubated with each composite resin for 24 h at 37 °C under orbital shaking (150 rpm). At the end, the cell suspension was collected and centrifuged for 10 min at 1000 × g and 4 °C. The free hemoglobin in the supernatant was quantified by the measurement of the absorbance at 540 nm.

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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Antifungal activity of dental resins containing amphotericin B-conjugated nanoparticles
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