Peripheral tetra-cationic Pt(II) porphyrins photo-inactivating rapidly growing mycobacteria: First application in mycobacteriology.

In this manuscript, we report, for the first time, the photoinactivation evaluation of tetra-cationic porphyrins with peripheral Pt (II)-bpy complexes in the photodynamic inactivation (PDI) of rapidly growing mycobacterial strains (RGM). Two different isomeric Pt (II)-porphyrins were synthetized and applied. PDI experiments in the strains of Mycobacteroides abscessus subsp. Abscessus (ATCC 19977), Mycolicibacterium fortuitum (ATCC 6841), Mycobacteroides abscessus subsp. Massiliense (ATCC 48898), and Mycolicibacterium smegmatis (ATCC 700084) conducted with adequate concentration (without aggregation) of photosensitizers (PS) under white-light illumination for 90 min showed that the most effective PS significantly reduced the concentration of viable mycobacteria. The present results show that positively charged porphyrins at the meta position (3-PtTPyP) are more efficient PS against M. abscessus, M. fortuitum, M. massiliense, and M. smegmatis. The effectiveness of the molecule as PS for PDI studies is also clear with mycobacteria, which is strongly related with the porphyrin peripheral charge and coordination platinum (II) compounds and consequently their solubility in physiological media. Tetra-cationic PS may be promising anti-mycobacterial PDI agents with potential applications in medical clinical cases and bioremediation.

Metal center ion effects on photoinactivating rapidly growing mycobacteria using water-soluble tetra-cationic porphyrins.

Rapidly growing mycobacteria (RGM) are pathogens that belong to the mycobacteriaceae family and responsible for causing mycobacterioses, which are infections of opportunistic nature and with increasing incidence rates in the world population. This work evaluated the use of six water-soluble cationic porphyrins as photosensitizers for the antimicrobial photodynamic therapy (aPDT) of four RGM strains: Mycolicibacterium fortuitum, Mycolicibacterium smeagmatis, Mycobacteroides abscessus subs. Abscessus, and Mycobacteroides abscessus subsp. massiliense. Experiments were conducted with an adequate concentration of photosensitizer under white-light irradiation conditions over 90 min and the results showed that porphyrins 1 and 2 (M = 2H or ZnII ion) were the most effective and significantly reduced the concentration of viable mycobacteria. The present work shows the result is dependent on the metal-center ion coordinated in the cationic porphyrin core. Moreover, we showed by atomic force microscopy (AFM) the possible membrane photodamage caused by reactive oxygen species and analyzed the morphology and adhesive force properties. Tetra-positively charged and water-soluble metalloporphyrins may be promising antimycobacterial aPDT agents with potential applications in medical clinical cases and bioremediation.

Antimicrobial activity of water-soluble tetra-cationic porphyrins on Pseudomonas aeruginosa.

This manuscript presents the cytotoxicity, antimicrobial activity, antibiofilm preliminary properties, and associated therapy with commercial drugs using water-soluble tetra-cationic porphyrins against Pseudomonas aeruginosa. Two commercial tetra-cationic porphyrins were tested against a standard strain of P. aeruginosa 01 (PA01) in antibacterial activity assays under dark conditions and irradiated with white light for 120 min. Porphyrin 4-H2TMePor showed better antimicrobial activity and was chosen for further tests. Increased minimum inhibitory concentration was observed in the presence of reactive oxygen species, suggesting that photooxidation was mediated by the singlet oxygen production. In the time-kill curve assay, 4-H2TMePor inhibited bacterial growth in 90 min of irradiation. The checkerboard assay revealed synergistic interactions. Biofilms of the standard PA01 strain and three clinical isolates were formed. The biofilm destruction assay was more efficient for PA01, significantly reducing the biofilm biomass formed compared to the positive control. The associated treatment to destroy the biofilm potentiated a significant decrease in the biofilm biomass compared to the positive control. The photosensitizer did not damage human keratinocytes or mouse fibroblasts in the cytotoxicity assays, demonstrating the safety of using 4-H2TMePor. Atomic force microscopy indicated lower adhesion force, higher cell wall deformation, and higher dissipation energy in the treated control compared to untreated PA01. Given our findings, it is evident that water-soluble tetra-cationic porphyrins have excellent antimicrobial and a preliminary antibiofilm activity against Gram-negative bacteria, proving to be a potential photosensitizer for clinical use.

Photodynamic inactivation of different Candida species and inhibition of biofilm formation induced by water-soluble porphyrins.

BACKGROUND: Candida spp. is the main fungal genus related to infections in humans, and its treatment has become a challenge due to the production of biofilm and its resistance/multi-resistance profile to conventional antifungals. Antimicrobial photodynamic therapy stands out as a treatment characterized by a broad spectrum of antimicrobial action, being able to induce oxidative stress in pathogens, and porphyrins are photosensitizers with high selectivity to pathogens. Thus, this work aimed to analyze the photoinactivation of different species of Candida by two cationic (4-H2TMeP+ and 3-H2TMeP+) and one anionic (4-H2TPSP‒) porphyrins. MATERIALS AND METHODS: Microdilution assays were performed to determine the MIC100, with subsequent determination of MFC100. Determination of oxidative species was done through the use of scavengers, while biofilm morphological features were investigated using the atomic force microscopy. RESULTS: Cationic porphyrins were significantly efficient in inactivating Candida albicans and non-albicans species with 100% growth inhibition and fungicidal activity (MFC100/MIC100 ≤ 4.0). The cationic porphyrins were also able to interfere in Candida spp biofilm formation. The photo-oxidative mechanism activated by 3-H2TMeP+ in Candida spp. is concurrent with the production of singlet oxygen and oxygen radical species. In the AFM analysis, 3-H2TMeP+ was able to reduce yeast adhesion to the surface. CONCLUSIONS: Cationic porphyrins can photo-inactivate different species of Candida in both planktonic and biofilm-associated forms, and reduce the adhesion of these fungi to the surface.

Nanomolar effective report of tetra-cationic silver(II) porphyrins against non-tuberculous mycobacteria in antimicrobial photodynamic approaches.

We report the photoinactivation evaluation of Ag(II) porphyrins (cationic AgTMeP and anionic AgTPPS) in the antimicrobial photodynamic therapy (aPDT) of rapidly growing mycobacterial strains. The aPDT assays in the Mycolicibacterium fortuitum, Mycobacteroides abscessus subs. abscessus, Mycobacteroides abscessus subsp. massiliense, and Mycolicibacterium smegmatis strains conducted without aggregating photosensitizers (PS) under irradiation for 90 min (270 J/cm2) showed that the most effective PS (nanomolar range) significantly reduced the concentration of viable mycobacteria. Structural damage on the Mycolicibacterium smegmatis non-pathogenic model was observed using atomic force microscopy, revealing that Ag(II)-porphyrin induced extensive changes in its electrical and adhesive forces, demonstrating changes in topography that may be linked to the action of different fractions of reactive oxygen species. The results presented in this paper provide solid evidence for using cationic porphyrin AgTMeP as an alternative to the conventional treatment of cutaneous mycobacteriosis and the disinfection of prosthetic devices and hospital equipment.

Simulation of CAD/CAM milling on lithium disilicate: Mechanical and topographic analyses of surface grinding different protocols.

The aim of this study was to evaluate the topography and the fatigue performance of lithium disilicate glass-ceramic after surface grinding through different laboratory protocols used to simulate the Computer-aided design/Computer-aided manufacturing (CAD/CAM) milling. Ceramic discs (IPS e.max CAD, Ø = 13.5 mm × 1.2 mm of thickness) were produced through different methodologies: milling in CAD/CAM system (CAD/CAM group); produced in-lab with a polished surface (POL group); or produced through in-lab methods and randomly distributed into five groups according to different grinding protocols to simulate the CAD/CAM milling [grinding with a CAD/CAM bur coupled to a mandrel (CAD/CAM Bur group); fine diamond bur using oscillatory movements (DBO group); fine diamond bur in x and y axes of the disc (DBXY group); #60-grit silicon carbide sandpaper (SiC group); and #60-grit wood sandpaper (WS group)]. The specimens were fatigue tested (n = 15) according to the step-stress method (initial load: 60 N; step-size: 20 N; 10,000 cycles/step; 20 Hz frequency). A roughness analysis was performed on all specimens, while fractal dimension (FD) and fractography were performed on representative samples. The Kaplan-Meier analysis showed that the POL (293.3 N) group presented better fatigue performance (higher load and number of cycles for failure) (p < 0.05) than the other groups (CAD/CAM = 222.7 N; CAD/CAM Bur = 181.3 N; DBO = 184.0 N; DBXY = 192.0 N; SiC = 182.6 N; WS = 182.6 N). For roughness, only the SiC (Ra = 1.616; Rz = 10.465) and WS (Ra = 1.673; Rz = 10.655) groups produced statistically similar Ra (µm) and Rz (µm) values to the CAD/CAM (Ra = 1.628; Rz = 9.571) group (p > 0.05). The surface created by CAD/CAM milling and POL group exhibited more complexity (FD) higher values than the experimental groups. For the ceramic surface topography images, the CAD/CAM milling visibly produced a uniform surface compared to the other groups; however, the POL group was the smoothest. The DBO, DBXY, SiC, and WS groups resulted in similar characteristics of surface topography. Therefore, although the SiC and WS groups showed similar roughness to the control group (CAD/CAM), no in-lab simulation method was fully capable to mimic the mechanical performance of the CAD/CAM-milled lithium disilicate glass-ceramic.

Photo-damage promoted by tetra-cationic palladium(II) porphyrins in rapidly growing mycobacteria.

Antimicrobial photodynamic therapy (aPDT) has gained prominence in microbiology, especially in treating non-invasive infections. Diseases such as mycobacteriosis, which causes localized infections and has a slow treatment, tend to be future targets for this type of technology. Therefore, this study aimed to explore the action of two isomeric Pd(II)-porphyrins on fast-growing mycobacterial strains (RGM). Tetra-cationic porphyrins (4-PdTPyP and 3-PdTPyP) were synthesized and applied against standard strains of Mycobacteroides abscessus subsp. abscessus (ATCC 19977), Mycolicibacterium fortuitum (ATCC 6841), Mycolicibacterium smegmatis (ATCC 700084), and Mycobacteroides abscessus subsp. massiliense (ATCC 48898). Reactive oxygen species (ROS) scavengers were used in an attempt to determine possible ROS produced by the photosensitizers (PS) under study. Moreover, the impact of porphyrin on the mycobacterial surface was further evaluated by atomic force microscopy (AFM), and we observed significant damage on cells walls and altered nanomechanical and electrostatic adhesion properties. The results presented herein show that the positively charged porphyrin at the meta position (3-PdTPyP) was the most efficient PS against the RGM strains, and its bactericidal activity was proven in two irradiation sessions, with singlet oxygen species being the main ROS involved in this process. This study demonstrated the therapeutic potential of porphyrins, especially the 3-PdTPyP derivative.

Microstructure, topography, surface roughness, fractal dimension, internal and marginal adaptation of pressed and milled lithium-disilicate monolithic restorations.

PURPOSE: To characterize the effect of two processing techniques (pressing and CAD/CAM - Computer Aided Design/Computer Aided Machining) of lithium-disilicate (LD) based crowns on the microstructure, topography, roughness, fractal dimension, internal and marginal adaptation. METHODS: One-hundred identical preparations for monolithic crowns were made with dentin analogue material (G10 epoxy resin). One of the preparations was scanned and a monolithic crown in lithium-disilicate was planned in the CAD system. Fifty crowns were milled in a wax pattern and subjected to pressing (IPS e.max Press), while 50 crowns were machined at CAD/CAM (IPS e.max CAD) and posteriorly crystallized. Internal and marginal adaptation were assessed via replica technique at four manufacturing moments (Milled wax pattern; LDPRESS; Milled LDCAD; Crystallized LDCAD) and considering 5 regions (margin, cervico-axial angle, axial wall, axial-occlusal angle and occlusal wall). Complementary analysis considering microstructure and topography, roughness and fractal dimension were performed in Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). RESULTS: The processing technique resulted in different ceramic microstructure, topography, roughness and fractal dimension, whereas CAD/CAM lead to smoother, more homogeneous but more complex topography features (higher fractal dimension) in comparison to the pressing technique (P<0.05). Regarding marginal and internal fit, LDPRESS crowns showed to be more adapted at the margin, while LDCAD were more adapted at the occluso-axial angle; other regions were not statistically different (α=0.05). CONCLUSIONS: CAD/CAM and the pressing techniques for manufacturing LD crowns lead to completely different ceramic surface characteristics and affect crown adaptation at the margin and at occluso-axial angle.

Hydrofluoric acid concentrations: Effect on the cyclic load-to-failure of machined lithium disilicate restorations.

OBJECTIVES: To evaluate the effects of the etching with different hydrofluoric acid (HF) concentrations on the cyclic load-to-failure (CLf) of machined lithium disilicate crowns cemented to dentin analogue material. METHODS: Pairs of dentin analogue prosthetic preparations and lithium disilicate ceramic crowns with simplified and standardized designs were machined (n=18). The preparations were etched with 10% HF (60s), followed by primer application. The intaglio surface of the ceramic crowns was treated as follows: non-etched (control, CTRL); or etched for 20s with different HF concentrations - 3% (HF3), or 5% (HF5), or 10% (HF10). A silane coating was then applied onto the treated ceramic surfaces, and they were adhesively cemented to the preparations. To perform the fatigue tests (staircase approach), a hemispheric stainless-steel piston (Ø=40mm) applied cyclic loads in the center of the crowns under water (initial load: 720N; step-size: 70N; cycles: 500,000; frequency: 20Hz). Additionally, topographic, fractographic, and fractal analyses were carried out. The fatigue data were analyzed using the Dixon and Mood method. RESULTS: Although the topographic and fractal analyses depicted the action of HF etching altering the superficial complexity and topography, the preponderant topography pattern was established by machining on CAD/CAM. All groups showed similar CLf (in N) (CTRL=805.00±91.23; HF3=781.25±29.87; HF5=755.00±154.49; HF10=833.75±100.74). SIGNIFICANCE: Etching with different HF acid concentrations did not promote a deleterious effect on the cyclic load-to-failure of machined lithium disilicate crowns.