Superhydrophobic Tipped Antimicrobial Photodynamic Therapy Device for the In Vivo Treatment of Periodontitis Using a Wistar Rat Model.

Limited options exist for treatment of periodontitis; scaling and root planing (SRP) are not sufficient to eradicate P. gingivalis and the resulting inflammatory disease. Chlorhexidine (CHX), used as an adjuvant to SRP, may reduce bacterial loads but leads to pain and staining, while evidence for its efficacy is lacking. Antibiotics are effective but can lead to drug-resistance. The rising concern of antibiotic resistance limits the future use of this treatment approach. This study evaluates the efficacy of a novel superhydrophobic (SH) antimicrobial photodynamic therapy (aPDT) device as an adjuvant to SRP for the treatment of periodontitis induced in a Wistar rat in vivo model relative to CHX. The SH-aPDT device comprises an SH silicone rubber strip coated with verteporfin photosensitizer (PS), sterilized, and secured onto a tapered plastic optical fiber tip connected to a red diode laser. The superhydrophobic polydimethylsiloxane (PDMS) strips were fabricated by using a novel soluble template method that creates a medical-grade elastomer with hierarchical surface roughness without the use of nanoparticles. Superhydrophobicity minimizes direct contact of the PS-coated surface with bacterial biofilms. Upon insertion of the device tip into the pocket and energizing the laser, the device generates singlet oxygen that effectively targets and eliminates bacteria within the periodontal pocket. SH-aPDT treatment using 125 J/cm2 of red light on three consecutive days reduced P. gingivalis significantly more than SRP-CHX controls (p < 0.05). Clinical parameters significantly improved (p < 0.05), and histology and stereometry results demonstrated SH-aPDT to be the most effective treatment for improving healing and reducing inflammation, with an increase in fibroblast cells and extracellular matrix and a reduction in vascularization, inflammatory cells, and COX-2 expression. The SH-aPDT approach resulted in complete disease clearance assessed 30 days after treatment initiation with significant reduction of the periodontal pocket and re-formation of the junctional epithelium at the enamel-cementum junction. PS isolation on a SH strip minimizes the potential for bacteria to develop resistance, where the treatment may be aided by the oxygen supply retained within the SH surface.

Improving antimicrobial activity against endodontic biofilm after exposure to blue light-activated novel curcumin nanoparticle.

New therapies involving natural products and nanobiotechnology open additional perspectives to reduce endodontic infections. Curcumin is a natural polyphenol extracted from the dry rhizome of curcuma long Linn with therapeutic properties for application in nanobiotechnology and as a photosensitizer for photodynamic therapy. This study aimed to synthesize a novel polymeric nanoparticle of poly (lactic-co-glycolic acid) (PLGA) loaded with curcumin (NP+Cur), and evaluate its antimicrobial activity against endodontic biofilms. Additionally, its biocompatibility using oral keratinocytes was assessed. The polymeric NP+Cur was prepared by the nanoprecipitation method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were calculated for the three endodontic bacteria (Enterococcus faecalis, Streptococcus oralis and Actinomyces viscosus). Antibacterial activity of NP+Cur against single- and multispecies biofilm pre-formed on the botton 24-well plate and into dentin tubules of bovine teeth were evaluated by colony forming units and confocal laser scanning microscopy. The pre-irradiation time was 5 min followed by exposure to blue light-emitting diode at 450 nm for the photodynamic treatment. Cell viability using oral keratinocytes was assessed by Alamar Blue assay. MIC and MBC showed antibacterial activity of NP+Cur against endodontic bacteria. A treatment of pre-formed biofilms of endodontic bacteria with NP+Cur also significantly decreased bacterial viability. The concentration of 325 µg/mL of photoactivated NP+Cur was the one that most reduced the viability of the endodontic bacteria evaluated. Regarding biocompatibility, NP+Cur 325 µg/mL and pure nanoparticles showed a cell viability greater than 80%. The novel polymeric nanoparticles loaded with curcumin may be a promising adjunct use to treatment of endodontic infections.

Effect of curcumin-loaded photoactivatable polymeric nanoparticle on peri-implantitis-related biofilm.

Curcumin has been used as a photosensitizer (PS) for antimicrobial photodynamic chemotherapy (PACT). However, its low solubility, instability, and poor bioavailability challenge its in vivo application. This study aimed to synthesize curcumin-loaded polymeric nanoparticles (curcumin-NP) and determine their antimicrobial and cytotoxic effects. Nanoparticles (NP) were synthesized using polycaprolactone (PCL) as a polymer by the nanoprecipitation method. Curcumin-NP was characterized by particle size, polydispersity index and zeta potential, scanning electron microscopy, and curcumin encapsulation efficiency (EE). Curcumin-NP was compared to free curcumin solubilized in 10% DMSO as photosensitizers for PACT in single and multispecies Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus oralis biofilms. Chlorhexidine 0.12% (CHX) and ultrapure water were used as positive and negative controls. The cytotoxic effect of curcumin-NP was evaluated on human periodontal ligament fibroblast cells (HPLF). Data were analyzed by ANOVA (α=0.05). Curcumin-NP exhibited homogeneity and stability in solution, small particle size, and 67.5% EE of curcumin. Curcumin-NP presented reduced antibiofilm activity at 500 µg/ml, although in planktonic cultures it showed inhibitory and bactericidal effect. Curcumin-NP and curcumin with and without photoactivation were not cytotoxic to HPLF cells. Curcumin-NP has antimicrobial and antibiofilm properties, with better effects when associated with blue light, being a promising therapy for preventing and treating peri-implant diseases.

Biosynthesis of silver nanoparticles from Syzygium cumini leaves and their potential effects on odontogenic pathogens and biofilms.

This study analyzed the antimicrobial and antibiofilm action and cytotoxicity of extract (HEScL) and silver nanoparticles (AgNPs-HEScL) from Syzygium cumini leaves. GC-MS, UV-Vis, EDX, FEG/SEM, DLS and zeta potential assays were used to characterize the extract or nanoparticles. Antimicrobial, antibiofilm and cytotoxicity analyses were carried out by in vitro methods: agar diffusion, microdilution and normal oral keratinocytes spontaneously immortalized (NOK-SI) cell culture. MICs of planktonic cells ranged from 31.2-250 (AgNPs-HEScL) to 1,296.8-10,375 µg/ml (HEScL) for Actinomyces naeslundii, Fusobacterium nucleatum, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus oralis, Veillonella dispar, and Candida albicans. AgNPs-HEScL showed antibiofilm effects (125-8,000 µg/ml) toward Candida albicans, Streptococcus mutans and Streptococcus oralis, and Staphylococcus aureus and Staphylococcus epidermidis. The NOK-SI exhibited no cytotoxicity when treated with 32.8 and 680.3 µg/ml of AgNPs-HEScL and HEScL, respectively, for 5 min. The data suggest potential antimicrobial and antibiofilm action of HEScL, and more specifically, AgNPs-HEScL, involving pathogens of medical and dental interest (dose-, time- and species-dependent). The cytotoxicity of HEScL and AgNPs-HEScL detected in NOK-SI was dose- and time-dependent. This study presents toxicological information about the lyophilized ethanolic extract of S. cumini leaves, including their metallic nanoparticles, and adds scientific values to incipient studies found in the literature.

Dramatic destruction of methicillin-resistant Staphylococcus aureus infections with a simple combination of amoxicillin and light-activated methylene blue.

BACKGROUND: Staphylococcus aureus is and continues to be a leading cause of bacterial infections throughout the world. Given the global dissemination of multi-drug resistant (MDR) S. aureus, particularly methicillin-resistant S. aureus (MRSA), novel solutions against S. aureus infections are urgently needed. In our study on the interactions between commonly used photosensitizers and antibiotics in the clinic, we discovered that MRSA can be dramatically destroyed by a simple combination of amoxicillin and light-activated methylene blue (MB). METHODS: To guide the clinical application of this combination therapy, we quantitatively assessed the interaction between light-activated MB and amoxicillin against S. aureus and its treatment order, dosage, and time length dependence. Furthermore, we evaluated the efficacy of this combination therapy in treating and halting the progression of MRSA infections with the catheter biofilm infection model and the pig skin burn infection model. In the end, we disclosed the antimicrobial mechanisms of this combination therapy to further facilitate its clinical translation. RESULTS: Amoxicillin and light-activated MB can mutually boost each other's uptake in S. aureus, producing up to 8 logs of reduction of MRSA infections when they are co-administrated. Such an anti-S. aureus synergy could be triggered with the currently used MB and amoxicillin clinical administration regimens. It is effective against S. aureus pathogens regardless of their antibiotic resistance backgrounds and does not create significant bacterial resistance with five days of continuous applications. It can lead to more than 99% of reduction of S. aureus infections established not only on the medical devices but also on the body surfaces. CONCLUSIONS: Possessing a fusion of effectiveness, safety, sustainability, and broad applicability, this simple combination of light-activated MB and amoxicillin can ultimately reform our treatment against MDR S. aureus pathogens including MRSA, significantly alleviating the health and economic burden of S. aureus infections across the world.

Evaluation of photosensitizer-containing superhydrophobic surfaces for the antibacterial treatment of periodontal biofilms.

Antimicrobial photodynamic therapy (aPDT) is a promising approach to control biofilms involved in periodontal diseases. However, certain challenges, such as staining of teeth, preferential interaction of photosensitizer (PS) with Gram-positive versus Gram-negative bacteria, and insufficient oxygen in hypoxic periodontal pockets have presented barriers to its use in the clinic. To overcome these challenges, a novel superhydrophobic (SH) film that generates airborne singlet oxygen has been developed. The SH-aPDT approach isolates the PS onto a topologically rough solid SH film on which channels allow air to diffuse to the PS surface, thus ensuring sufficient oxygen supply. Upon illumination, gas phase singlet oxygen (1O2) is produced and diffuses from the SH surface to the underlying biofilm. The killing efficacy was assessed as a function of transmitted fluence (17.9-89.5 J/cm2) and chorin e6 loading (96-1110 nmol/cm2) by counting of colony forming units, biofilm metabolism by XTT and confocal microscopy. The decrease in viability of both Gram-positive and Gram-negative bacteria in a multi-species biofilm was found to be linearly dependent on the fluence as well as the loading of the PS up to 71.6 J/cm2 when 1110 nmols/cm2 of chlorin e6 was used. A > 4.6 log bacterial reduction was observed under these conditions (p < 0.05). This novel SH-aPDT approach shows promise as an effective method to disinfect multi-species bacterial biofilms associated with periodontal disease and will be evaluated in animal models in future studies.

Antimicrobial Photodynamic Inactivation Using Topical and Superhydrophobic Sensitizer Techniques: A Perspective from Diffusion in Biofilms.

This review describes nanoparticle and dye diffusion in bacterial biofilms in the context of antimicrobial photodynamic inactivation (aPDI). aPDI requires the diffusion of a photosensitizer (Sens) into the biofilm and subsequent photoactivation of oxygen for the generation of reactive oxygen species (ROS) that inactivate microbes. Molecular diffusion in biofilms has been long investigated, whereas this review is intended to draw a logical link between diffusion in biofilms and ROS, a combination that leads to the current state of aPDI and superhydrophobic aPDI (SH-aPDI). This review should be of interest to photochemists, photobiologists and researchers in material and antimicrobial sciences as is ties together conventional aPDI with the emerging subject of SH-aPDI.

Antimicrobial effects of silver nanoparticles and extracts of Syzygium cumini flowers and seeds: Periodontal, cariogenic and opportunistic pathogens.

OBJECTIVE: This study aimed to analyze the antimicrobial effects of lyophilized hydroalcoholic extract (HEScSeed and HEScFlower) and silver nanoparticles (AgNPs-HEScSeed and AgNPs-HEScFlower) of S. cumini seed and flower, and to characterize some compounds of these extracts and their NPs. DESIGN: Phytochemical screening was performed by GC-MS. Nanoparticles were characterized by UV-vis spectroscopy, energy-dispersive X-ray (EDX) spectrophotometry, scanning electron microscopy (SEM) and field emission gun (FEG), dynamic light scattering (DLS) and zeta potential (ZP). Antimicrobial susceptibility tests were analyzed by broth microdilution and agar diffusion methods. RESULTS: HEScSeed and HEScFlower showed 7 and 17 phytochemical compounds, respectively. AgNPs-plant extracts were reported as stable and with variable shapes and sizes. All studied species (A. naeslundii, C. albicans, F. nucleatum, S. aureus, S. epidermidis, S. mutans, S. oralis and V. dispar) were susceptible to extracts and AgNPs-plant extracts, with varying degrees of antimicrobial activities (extract: 648.4-5,187.5 µg/mL; AgNPs-plant: 31.2-2,000 µg/mL). CONCLUSION: The extracts of S. cumini seed and flower have antimicrobial action against pathogens of medical and dental interest, whose MIC and MMC are species-dependent. The AgNPs-HEScSeed and AgNPs-HEScFlower have different shapes, sizes, organic compounds, stability and electronegativity (capping), characteristics that contribute to their bacteriostatic and fungistatic effects, but at significantly lower concentrations than plant extracts.

Terpinen-4-ol and nystatin co-loaded precursor of liquid crystalline system for topical treatment of oral candidiasis.

This study was performed to develop a liquid crystalline system (LCS) incorporated with terpinen-4-ol and nystatin to evaluate its antifungal, antibiofilm, and synergistic/modulatory activity against Candida albicans. The LCS was composed of a dispersion containing 40% propoxylated and ethoxylated cetyl alcohol, 40% oleic acid, and 0.5% chitosan dispersion. According to analysis by polarized light microscopy, rheology, and mucoadhesion studies, the incorporation of 100% artificial saliva increased the pseudoplasticity, consistency index, viscosity, and mucoadhesion of the formulation. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity; the LCS containing terpinen-4-ol and nystatin effectively inhibited C. albicans growth at a lower concentration, displaying a synergistic action. Therefore, LCS incorporated with terpinen-4-ol and nystatin is a promising alternative for preventing and treating infections and shows potential for the development of therapeutic strategies against candidiasis.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans.

Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans

Abstract Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Interactions between Terpinen-4-ol and Nystatin on biofilm of Candida albicans and Candida tropicalis.

The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.

Antimicrobial effects of terpinen-4-ol against oral pathogens and its capacity for the modulation of gene expression.

This study evaluated the antibacterial activity of terpinen-4-ol against Streptococcus mutans and Lactobacillus acidophilus and its influence on gbpA (S. mutans) and slpA (L. acidophilus) gene expression. As measured by XTT assay, the concentrations of terpinen-4-ol that effectively inhibited the biofilm were 0.24% and 0.95% for S. mutans and L. acidophilus, respectively. Confocal microscopy revealed the presence of a biofilm attached to the enamel and dentin block surfaces with significant terpinen-4-ol effects against these microorganisms. The expression of the gbpA and slpA genes involved in adherence and biofilm formation was investigated using RT-PCR. Expression of these genes decreased after 15 min with 0.24% and 0.95% terpinen-4-ol in S. mutans and L. acidophilus, respectively. These findings demonstrate the antimicrobial activity of terpinen-4-ol and its ability to modulate the expression of gbpA and slpA genes, emphasizing the therapeutic capacity of terpinen-4-ol as an alternative to inhibit adherence in biofilm.

In vitro antibacterial and cytotoxic activities of carvacrol and terpinen-4-ol against biofilm formation on titanium implant surfaces.

This study evaluated the antibacterial properties of carvacrol and terpinen-4-ol against Porphyromonas gingivalis and Fusobacterium nucleatum and its cytotoxic effects on fibroblast cells. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were examined. The minimum biofilm inhibition concentration (MBIC) was evaluated by XTT assay. Biofilm decontamination on titanium surfaces was quantified (CFU ml-1), evaluated by confocal laser scanning microscopy (CLSM) and cytotoxic activity by MTT. The MIC and MBC for carvacrol were 0.007% and 0.002% for P. gingivalis and F. nucleatum, and 0.06% for terpinen-4-ol for both microorganisms. The MBIC for carvacrol was 0.03% and 0.06% for P. gingivalis and F. nucleatum, and for terpinen-4-ol was 0.06% and 0.24%. The results indicated anti-biofilm activity using carvacrol (0.26%, 0.06%) and terpinen-4-ol (0.95%, 0.24%) and showed cytotoxic activity similar to chlorohexidine (CHX). However, terpinen-4-ol (0.24%) showed higher cell viability than other treatments. Carvacrol and terpinen-4-ol showed antibacterial activity in respect of reducing biofilms. Moreover, CHX-like cytotoxicity was observed.

Interactions between Terpinen-4-ol and Nystatin on biofilm of Candida albicans and Candida tropicalis

Abstract The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.

Resumo O objetivo desse estudo foi avaliar a atividade antifúngica do Terpinen4-ol associado à nistatina em biofilmes simples e misto, formados por Candida albicans e Candida tropicalis, bem como o efeito do terpinen-4-ol na adesão em células orais e atividade enzimática. As concentrações inibitórias mínimas e as concentrações fungicidas mínimas do terpinen-4-ol e da nistatina em Candida albicans e Candida tropicalis foram determinadas pelo método de microdiluição em caldo, juntamente com a atividade sinérgica (método do tabuleiro de "xadrez"). Biofilmes simples e misto foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (CFU/mL). O efeito do Terpinen-4-ol na adesão de Candida albicans e Candida tropicalis em co-cultura com queratinócitos orais (NOK Si) foi avaliado, bem como a atividade enzimática, medindo o tamanho da zona de precipitação, após o crescimento em ágar fosfolipase, protease e hemolisina. O terpinen-4-ol (4.53 mg mL-1) e a nistatina (0,008 mg mL-1) conseguiram inibir o crescimento de biofilmes e um efeito antifúngico sinérgico foi demonstrado com a associação de fármaco, reduzindo a concentração inibidora de nistatina até 8 vezes em biofilme simpes de Candida albicans e 2 vezes em biofilme misto. Uma pequena diminuição na adesão de Candida tropicalis em células NOK Si foi mostrada após o tratamento com terpinen-4-ol e a nistatina teve um efeito maior para ambas as espécies. Para a atividade enzimática, as drogas não apresentaram ação. O efeito potencializado pela combinação de terpinen-4-ol e nistatina e a redução de adesão evidenciam seu potencial como agente anti-fúngico.