Anti-dermatophyte activity of Ti[O.sub.2] NPs colloidal prepared by pulsed laser ablation in liquid environment.
Dermatophytes are a pathogenic filamentous fungi such as T. rubrum that cause essential superficial infections (Dermatophytosis) and have the ability to infestation keratinized tissues such as skin, hair and nails . They most usually infect the feet, axillae, scalp, and nails with a different severity of infection . The major structural of the human skin and nails is keratin, a fibrous protein. The fungal pathogen has the ability to produce proteolytic enzymes so T. rubrum invades the human skin and nail via keratin fragmentation]. Onychomycosis is a fungal infection of the fingernails or toenails caused by a variety of fungi including dermatophytes such as T. rubrum in this case the fungus invades the hyponychium at the distal lateral part of the nail bed or arise from under the nail fold and causes discoloration, thickening, and detachment from the nail bed and it is possible to expands to tinea pedis. . Onychomycosis is more common in older adults and occur in human that suffers from immunodeficiency [5, 6]. With the evolution of biomedical nanomaterials, new antimicrobial was developed due to the physiochemical properties of NPs . NPs usually ranging in dimension from 1-100 nanometers (mn) so they have unique properties from bulk particle that has the possibility of controlling and deal with the structures at molecular and atomic level. The percentage of atoms at the surface of NPs more than the total number of atoms at the surface of bulk particles so the surface -to-volume ratios of NPs become large. The physiochemical properties of NPs are varied in nature and they have highly viable in biomedical field and antimicrobial agent . The antimicrobial activity and physicochemical properties of NPs against biological molecules were related to their small size, high surface area to volume ratio, solubility, shape, surface coatings and charge. As well as the biological molecules sizes are similar to the NPs structures that lead to easy penetration of NPs inside the microorganisms (i.e small particle size and high surface area of NPs enhance their interaction with microorganisms) [9,10], Titanium dioxide NPs (TiCT NPs) is a promising material, used in many applications due to its high photo catalytic activity, dielectric properties, high stability and low cost [11,12,13], TiC[O.sub.2] NPs have different chemical, magnetic, optical and structural properties and they have more toxicity effects than its bulk particles so it was used in pharmaceutical products, catheters to prevent urinary tract infections, cosmetics, dental implants and packaging. Photoactivity of Ti[O.sub.2] NPs was effective against Gram-positive bacteria, Gram-negative bacteria, fungi, and bacteriophage. The studies have shown that the cell membranes that is exposed to Ti[O.sub.2] NPs will destroy followed by cell wall damage leading to the cell death . In comparison to the conventional antibiotics, nanostructured antimicrobial is regarded assistant factor in reducing the toxicity and lowering the cost more stable for long-term storage so NPs can resist high pH and temperature without being inactivated , NPs production by pulsed laser ablation of targets in liquids has lately become a promising technique for several advantages such as, straightforward, ease of production, NPs synthesizing in liquid avoiding the use of vacuum apparatus, direct synthesizing of NPs in solutions, pure colloidal solutions without the formation of contamination (green technique), all particles are collected by one-step and low costs of processing ,
MATERIALS AND METHODS
Titanium plate was supplied from Danyang Xinli Alloy company, China. The purity of the plate was examined by using Energy dispersive -X ray fluorescence (ED-XRF) device (model XEPOS) and it was about 98.4% as shown in Tablel and Fig. 1. The Titanium plate was cut to square-shaped plates with 1cm x 1cm x 0.1 mm dimensions. Each plate was cleaned by ethanol and double distilled de-ionized water and polished with emery paper to be ready for NPs fabrication process.
Preparation of Ti[O.sub.2] NPs colloidal:
Titanium NPs colloidal was prepared by using pulsed laser ablation (PLA) in liquid enviromnent. Titanium plate with 1cm x 1cm x 0.1 mm dimensions was fixed at the bottom of cylindrical glass vessel filled with 5ml from DDDW. The immersed plate was focused by Q-switched pulsed Nd:YAG laser at room temperature. The details of the laser beam parameters are summarized in Table. 2 and the schematic diagram of the experimental set up is shown in Fig. 2.
Isolate of T. rubrum:
T. rubrum was diagnosed in Biological Postgraduate Lab/Collage of Science/University of Baghdad.
Effect of Ti[O.sub.2] NPs on T. rubrum:
Standard (BDH-100mn) and prepared TiCf NPs were used to study their effects on viability of T. rubrum. The effect of Ti[O.sub.2] NPs was determined by direct exposme of (1 x [10.sup.5]) cell/ml of fungal cell suspension with different concentrations (100, 200 and 300) [micro]g/ml of standard Ti[O.sub.2] NPs and (37, 75 and 150) [micro]g/ml of prepared Ti[O.sub.2] NPs for 1, 2 and 3 hrs.
Sabouraud's dextrose agar (SDA) solution was prepared by dissolved 65mg/ml of SDA powder in 1000 ml of DDDW. 25ml of the prepared SDA was poured in each Petri-dishe and supplemented with (5[micro]g/ml) cycloheximide and (10 [micro]g/ml) amoxicillin for maintenance and growth of dermatophyte isolate.
Four holes (6 mm in diameter) were punched in each SDA Petri-dishe. One hole of each Petri-dishe was loaded with(1 x [10.sup.5]) cell/ml of fungal cell suspension (control group) and the other holes were loaded with the exposed fungal cell suspension (treated group) then incubated at 28[degrees]C for six days. The treated and untreated colonies diameters were measured at third and sixth day. The inhibition rate of T. rubrum (%) was expressed as follows:
Inhibition rate (%) = (Control - Test/Control) x 100
Control = Untreated colony diameter.
All data were recorded as M [+ or -] SD, and statistically analyzed using SPSS software version 16, ANOVAI with p [less than or equal to] 0.05 being Least Significant Differences LSD as well as descriptive analysis was used to calculate the percent of inhibition rate.
RESULTS AND DISCUSSION
Optical properties of Ti[O.sub.2] NPs colloidal:
Shape of the absorption spectrum, peak position, type, homogeneity and concentration of the synthesized Ti[O.sub.2] NPs colloidal were determined by UV-Visible absorption spectra of the solution by using UV-Vis spectrophotometer.
Effect of the number of laser pulses:
Fig. 3 shows The absorption spectra of Ti[O.sub.2] NPs colloidal. The beam spot diameter at the metallic surface was 1.1mm as shown in Fig. 4. The number of utilized pulses ranged from 300 to 700 pulses. The laser beam was focused on the immersed metal plate in DDDW. The energy of laser beam was absorbed by the metal surface and coupled with electrons in valence and conduction bands leading to breaking of electron bonds and the excited electrons transfer their energy to the other electrons and the lattice by electron-electron and electron- phonon collisions followed by rapid vaporization due to the heat from ionized atoms and electrons in the liquid thus high pressure and temperature plasma generated and the ionized electrons accelerate by collisions inside the plasma [17,18]. Strong shockwave was created and the plasma plume expanded adiabatically inside the confining liquid and mixed with the surrounding liquid . As a result, visible cloud of the metallic vapor was observed up the metal surface and Ti[O.sub.2] NPs colloidal formed and the solution changed to gray color. The nucleation, colloidal concentration, particle size distribution, density. and formation of Ti[O.sub.2] NPs in the liquid were enhanced by increasing laser pulses. The absorption spectra of Ti[O.sub.2] NPs peaked at 290nm.
Effect of the laser wavelengths:
The effect of laser wavelengths on the absorption spectra of Ti[O.sub.2] NPs was studied by using two wavelengths; 1064nm and 532nm respectively, as shown in Fig. 5. The fundamental wavelength (1064 nm) and the second harmonic generation (532 nm) of Nd:YAG laser were applied with energy of 490 mJ, 700 pulse and 6 Hz repetition rate. The laser was focused at the immersed titanium plate in 5ml of DDDW and the spot diameter at the metal surface was 0.8mm as shown in Fig. 6. The plasma formed upon the metal surface as a visible cloud of metal vapor. The gray colloidal solution of Ti[O.sub.2] NPs was obtained when the number of laser pulses was increased. The color of solution was changed faster for the laser wavelength of 1064 nm than that 532nm.
The plasma temperature generated by 1064 nm was higher than that generated by 532 nm due to the strong inverse Bremsstrahlung at the IR region so the amount of extracted metal from the metal surface at 1064 nm was higher compared to 532 nm leading to increase in the ablation efficiency . Increase in the NPs concentration was observed at 1064nm followed by rise in the peak of absorption spectrum. On the contrary the ablation efficiency reduced at the green wavelength (532 nm) due to photo-fragmentation process where the high photons energy were absorbed by NPs colloidal, consequently the amount of photons that were focused at the immersed titanium plate reduce .
Particle size analyzer:
Particle size distribution was investigated using Particle Size Analyzer. The intensity distribution of particle size was inhomogeneous and the particle size diameter ranging from 33.8 to 200nm as shown in Fig. 7.
Negative value of zeta potential (-14.81mv) was obtained from the produced Ti[O.sub.2] NPs colloidal as shown in Fig. 8. The negative surface charge of the solution was attributed to the high pH where H+ release out to the surface thus negative zeta potential is produce .
Morphologies of T1[O.sub.2] NPs:
Transmission Electron Microscopy (TEM):
Particle size distribution and morphology of Ti[O.sub.2] NPs were characterized by Transmission Electron Microscopy (TEM). The crystalline shape of the generated Ti[O.sub.2] NPs by fundamental wavelength (1064 nm) and second harmonic generation (532 nm) of Q-switched pulsed Nd:YAG laser was spherical and the particle size distribution ranged from 30 to 110 nm at 1064 nm and ranged from 10 to 90 nm at 532 nm as shown in Fig. 9 and Fig. 10, respectively.
Atomic Force Microscopy (AFM):
Fig. 11 a and b show 2-D and 3-D surface morphologies AFM photos of pure Ti[O.sub.2] NPs prepared by 1064nm Q-switched pulsed ND:YAG laser and c shows the granularity distribution chart. The ablation process was carried out by applied 700 pulse of laser beam operated at 490 mJ/pulse with repetition rate of 6 Hz. The average diameter of produced NPs was 103.35 nm.
Antifungal activity of Ti[O.sub.2] NPs:
Antifungal activity of Ti[O.sub.2] NPs was studied against dermatophyte T. rubrum where different concentrations of Ti[O.sub.2] NPs were tested on the cell suspension. The antifungal activity of various concentrations like (37, 75 and 150) [micro]g/ml of prepared and (100, 200 and 300) [micro]g/ml of standard Ti[O.sub.2] NPs was observed in the fungal colonies diameters growth as shown in Tables 3 and 4, respectively. The slow growth in the fungal colonies diameters that their cells were exposed to Ti[O.sub.2] NPs was observed at different exposure times and different incubation times, on the contrary the fungal colonies that their cells were not exposed to Ti[O.sub.2] NPs (control groups) naturally grew fast as shown in Fig. 12. The colony diameter growth was affected by two factors; These factors were the concentration of the NPs and the exposure times. Increase in the concentration of Ti[O.sub.2] NPs leading to decrease in the colony diameter growth this means that the Ti[O.sub.2] NPs activity increase with increasing Ti[O.sub.2] NPs concentration. Increased exposure time of Ti[O.sub.2] NPs with fungal cell suspension before their culture in the fungal culture media also caused decreasing in the colonies diameters growth so the growth of the fungal colonies diameters that their cells were exposed for 3 hrs with Ti[O.sub.2] NPs was slower than the growth of the fungal colonies diameters that their cells were exposed for 2 and 1 hrs. The inhibition rate of T. rubrum increased when the concentration of Ti[O.sub.2] NPs and exposure time increased as shown in Figs 14 and 15.
The antimicrobial activity of Ti[O.sub.2] NPs attributed to their small size (NPs size is not much different from the biomolecules size), morphology, high surface area to volume ratio, solubility, shape, surface coatings and charge. Reactive oxidative Stress (ROS) are reactive oxygen species that caused by chemical reactions between NPs and biomolecules with high reactive activity. ROS contain superoxide radical ([O.sup.-.sub.2]), hydrogen peroxide and hydroxyl radicals that can react with biomolecules and induce an imbalance between the reactive oxygen and the biological system's efficiency to detoxify leading to disruption of the cell wall. Excessive abundance of oxidative stress may also defect in the protein, enzyme, lipids and nucleic acids which further leads to cell death and inhibition or mutations in DNA[11,8].
Results confirmed that the two wavelengths (1064nm and 532nm) of Q-switched Nd:YAG laser technique have the potential to produce Ti[O.sub.2] NPs with a peak of absorption spectrum in 290nm, spherical particles shape, suitable particle size (30-110 nm at 1064nm and 10- 90 nm at 532nm) and negative zeta potential (-14.81mv). In vitro study demonstrated that these Ti[O.sub.2] NPs have potential effect on viability of T. rubrum via increasing their inhibition rate, significantly at highest concentration (150 [micro]g/ml) and highest exposure time (3 hrs).
Thanks and high appreciation is given to Laser and Optoelectronics Engineering Department and Environmental Research Center, University of Technology, Iraq, Baghdad for support this research.
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(1) A. Kadhim, (2) Azhar M. Haleem, (3) Ruaa H. Abass
(1) Laser and Optoelectronic Eng. Department, University of Technology (UOT), p.o 35256 Baghdad, Iraq.
(2) Environmental Research Center, University of Technology (UOT), Baghdad p.o 35256, Iraq.
(3) Laser and Optoelectronic Eng. Department, University of Technology (UOT), Baghdad p.o 35256 Iraq.
Address For Correspondence:
Ruaa H. Abass, Laser and Optoelectronic Eng. Department, University of Technology (UOT), p.o 35256 Baghdad, Iraq.
Received 12 August 2016; Accepted 17 December 2016; Available online 22 December 2016
Caption: Fig. 1: ED-XRF spectrum of Titanium plate
Caption: Fig. 2: Schematic diagram of the experimental set up
Caption: Fig. 3: The absorption spectra of Ti[O.sub.2] NPs colloidal at 700 pulse and 300 pulse, respectively, 6Hz,and [lambda]=1064 nm.
Caption: Fig. 4: Beam spot diameter of 1064nm laser beam
Caption: Fig. 5: The absorption spectra of Ti[O.sub.2] NPs colloidal at 1064 nm and 532nm, respectively, 6Hz, and 700 pulse.
Caption: Fig. 6: The spot diameter of the 532nm laser beam
Caption: Fig. 7: Particle size distribution of Ti[O.sub.2] NPs
Caption: Fig. 8: Zeta potential of Ti[O.sub.2] NPs colloidal
Caption: Fig. 9: a TEM image of Ti[O.sub.2] NPs b particle size distribution of Ti[O.sub.2] produced by ablating Ti plate in DDDW with 490 mj, 700 pulse, 1064 nm and 6Hz.
Caption: Fig. 10: a TEM image of Ti[O.sub.2] NPs b particle size distribution of Ti[O.sub.2] NPs produced by ablating Ti plate in DDDW with 490 mj, 700 pulse, 532nm and 6Hz.
Caption: Fig. 11: AFM of prepared Ti[O.sub.2] NPs by pulsed laser a 2D surface morphology b 3D surface morphology and c the granularity cumulation distribution chart.
Caption: Fig. 12: a The colonies diameters of control group and treated group of T. rubrum at different exposure times ([a.sub.1], [a.sub.2] and [a.sub.3]) after three days incubation time.
Caption: Fig. 13: b The colonies diameters of control group and treated group of T. rubrum at different exposure times ([b.sub.1], [b.sub.2] and [b.sub.3]) after six days incubation time.
Table 1: Titanium analysis by ED-XRF Z Symbol Element Concentration Abs. Error 12 Mg Magnesium < 0.027 % (0.0) % 13 Al Aluminum < 0.0060 % (0.0) % 14 Si Silicon < 0.0021 % (0.0) % 15 P Phosphorus < 0.0011 % (0.00052) % 16 S Sulfur < 0.0020 % (0.0) % 22 Ti Titanium 98.40 % 0.14 % 23 V Vanadium < 0.043 % (0.0) % 24 Cr Chromium < 0.0032 % (0.0) % 25 Mn Manganese < 0.0031 % (0.0022) % 26 Fe Iron 0.0585 % 0.0043 % 27 Co Cobalt 0.0052 % 0.0028 % 28 Ni Nickel 0.0101 % 0.0021 % 29 Cu Copper 0.0058 % 0.0015 % 30 Zn Zinc 0.0121 % 0.0016 % 33 As Arsenic 0.00364 % 0.00047 % 40 Zr Zirconium < 0.043 % (0.0) % 41 Nb Niobium < 0.0055 % (0.0) % 42 Mo Molybdenum 0.469 % 0.045 % 47 Ag Silver 0.0144 % 0.0078 % 48 Cd Cadmium 0.0093 % 0.0050 % 50 Sn Tin < 0.00035 % (0.0) % 51 Sb Antimony 0.0095 % 0.0095 % 74 W Tungsten < 0.0015 % (0.0) % 82 Pb Lead < 0.00090 % (0.0) % Table 2: The details of laser beam parameters of nanosecond pulsed Nd:YAG laser that used in this investigation. Laser parameters Details Wavelengths 1064 nm and 532 nm Laser energy 490 mj Repetition rate 6 Hz Spot diameter 1.1 mm for 1064 nm and 0.8 mm for 532 nm Pulse duration 10 ns Number of pulses 300-700 pulses Focal length 10cm Table 3: Colony diameter in centimeter of 1 x [10.sup.5] cell/ ml of T. rubrum treated with different concentrations of prepared Ti[O.sub.2] NPs at different exposure times and different incubation times. Times\Conc Three-days incubation time [micro]g/ml 1 hr exposure time 2 hrs exposure time 0.0 0.9 [+ or -] 0.03a 0.89 [+ or -] 0.03a 37 0.7 [+ or -] 0.04b 0.4 [+ or -] 0.045b 75 0.5 [+ or -] 0.02c 0.3 [+ or -] 0.025c 150 0.4 [+ or -] 0.03c 0.21 [+ or -] 0.02d Times\Conc Three-days Six-days incubation time [micro]g/ml incubation time 3 hrs exposure time 1 hr exposure time 0.0 0.91 [+ or -] 0.05a 2.46 [+ or -] 0.15a 37 0.4 [+ or -] 0.02b 1.33 [+ or -] 0.3b 75 0.3 [+ or -] 0.02c 1.03 [+ or -] 0.05c 150 0.2 [+ or -] 0.02d 0.36 [+ or -] 0.057d Times\Conc Six-days incubation time [micro]g/ml 2 hrs exposure time 3 hrs exposure time 0.0 2.06 [+ or -] 0.3a 2.76 [+ or -] 0.3a 37 0.95 [+ or -] 0.05b 0.43 [+ or -] 0.15b 75 0.33 [+ or -] 0.05c 0.33 [+ or -] 0.15c 150 0.26 [+ or -] 0.11c 0.23 [+ or -] 0.05d * Each number represent M [+ or -] SD of three replicate. * Various letters in each column represent significant differences at (p< 0.05). Table 4: Colony diameter in centimeter of 1 x [10.sup.5] cell/ ml of T. rubrum treated with different concentrations of standard Ti[O.sub.2] NPs at different exposure times and different incubation times. Times\Concx Three-days incubation time [micro]g/ml 1 hr exposure time 2 hrs exposure time 0.0 0.9 [+ or -] 0.005a 0.88 [+ or -] 0.02a 100 0.7 [+ or -] 0.1b 0.41 [+ or -] 0.01b 200 0.7 [+ or -] 0.05b 0.36 [+ or -] 0.017c 300 0.51 [+ or -] 0.02c 0.34 [+ or -] 0.03c Times\Concx Three-days Six-days incubation time [micro]g/ml incubation time 3 hrs exposure time 1 hr exposure time 0.0 0.92 [+ or -] 0.04a 2.3 [+ or -] 0.3a 100 0.41 [+ or -] 0.02b 1.26 [+ or -] 0.11b 200 0.33 [+ or -] 0.05c 1.16 [+ or -] 0.11b 300 0.33 [+ or -] 0.028c 1.13 [+ or -] 0.05b Times\Concx Six-days incubation time [micro]g/ml 2 hrs exposure time 3 hrs exposure time 0.0 2.36 [+ or -] 0.21a 2.53 [+ or -] 0.32a 100 1.16 [+ or -] 0.28b 1.13 [+ or -] 0.15b 200 1.13 [+ or -] 0.23b 1.06 [+ or -] 0.11c 300 1.1 [+ or -] 0.17b 1.03 [+ or -] 0.05b * Each number represents M [+ or -] SD of three replicate. * Various letters in each column represent significant differences at (p< 0.05). Fig. 14: Inhibition rate of treated T. rubrum with prepared Ti[O.sub.2] NPs at different concentrations (37,75 and 150) [micro]g/ml and exposure times (1 hr, 2hrs and 3 hrs) (a). Three days incubation time (b). Six days incubation time. a Concentration ([micro]g/ml) 37 75 150 1 hr 22 55 56 2 hrs 44 66 67 3 hrs 55 77 78 b Concentration ([micro]g/ml) 37 75 150 1 hr 45.9 53.8 84 2 hrs 58 83.9 88 3 hrs 85 87 91.6 Note: Table made from bar graph. Fig. 15: Inhibition rate of treated T. rubrum with standard Ti[O.sub.2] NPs at different concentrations (37,75 and 150) [micro]g/ml and exposure times (1 hr, 2hrs and 3 hrs) (a). Three days incubation time (b). Six days incubation time. a Concentration ([micro]g/ml) 100 200 300 1 hr 22 53 55 2 hrs 22 59 64 3 hrs 43 61 64 b Concentration ([micro]g/ml) 100 200 300 1 hr 45 50 55 2 hrs 49 52 58 3 hrs 52 52 59 Note: Table made from bar graph.