The E ﬀ ect of Antimicrobial Photodynamic Therapy Using Chlorophyllin–Phycocyanin Mixture on Enterococcus faecalis : The Inﬂuence of Di ﬀ erent Light Sources

: The purpose of this study was to evaluate the in vitro e ﬀ ect of the chlorophyllin–phycocyanin mixture (Photoactive + ) as a photosensitizer (PS) during antimicrobial photodynamic therapy (aPDT) on the count of Enterococcus faecalis ( E. faecalis ) using di ﬀ erent light sources. The antimicrobial e ﬀ ect of aPDT with chlorophyllin–phycocyanin mixture using di ﬀ erent light sources including diode laser ( λ = 660 nm), diode laser ( λ = 635 nm), LED ( λ = 450 ± 30 nm) alone or in combination was assessed using microbial cell viability assay against E. faecalis . In addition, the cell cytotoxicity of Photoactive + was assessed on human gingival ﬁbroblast (HuGu) cells by MTT assay; E . faecalis growth when treated by both red wavelengths (635 nm, 660 nm) and combination of LED (420–480 nm) and red wavelengths (635 nm, 660 nm), signiﬁcantly reduced compared to the control group ( p < 0.05). There was no signiﬁcant reduction in the number of viable cells exposed to Photoactive + compared to the control group ( p < 0.05). This study shows that the application of chlorophyllin–phycocyanin mixture and irradiation with emission of red light achieved a better result for bacterial count reduction, compared to a control. This component can be applied safely due to very negligible cytotoxicity.


Introduction
The complete elimination of microorganisms from root canal space is mandatory to increase the success rate of endodontic treatment [1]. Among different bacteria found in the root canal, Enterococcus faecalis (E. faecalis) has significant implication in secondary endodontic infections, due to its tolerance of harsh conditions [2]. Bacterial reduction within the root canal system is commonly attempted through the use of mechanical preparation and chemical irrigants [3] and adjunctive use of high and low power laser irradiance may be employed to enhance the results [4]. Among low power methods, antimicrobial photodynamic therapy (aPDT) is based on the application of a photosensitizer (PS) which, when irradiated by an appropriate wavelength result in the generation of reactive oxygen species (ROS) and other free radicals which are toxic for microorganisms which finally cause cell death [5,6].
Different PSs are used for aPDT in endodontic therapy including methylene blue (MB), toluidine blue (TBO) and indocyanine green (ICG), all of which can be activated by a commercially available diode laser of specific complementary wavelength [7,8]. Chiniforush et al. evaluated biofilm formation gene expression (esp) in E. faecalis treated using MB, TBO and ICG accompanied by 660, 635 and 810 nm diode laser irradiation wavelengths, respectively. They concluded that ICG demonstrated higher efficacy in the reduction of esp expression and can be considered as the best PS in treating endodontic infection [9]. The application of a natural product for treatment and management of disease has gained special attention [10].
Photoactive+ is a mixture of chlorophyllin-phycocyanin that is effective against biofilms of Gram-positive and Gram-negative bacteria, when activated by red (620-660 nm) and blue (400-450 nm) photonic energy wavelengths [11]. chlorophyllin is used as a coloring agent in food industry with several characteristics including antimicrobial and antitumoral effects [12] and phycocyanin extracted from Spirulina as an FDA-approved agent for coloring food is a water-soluble nontoxic agent with anticancer, antioxidant, antimicrobial and anti-inflammatory effects [13]. These natural products possess pharmacological activity with no toxic side effects [14]. The combined application of these two natural products with photo-activation using separate light sources may enhance the results.
The aim of this study was to evaluate the effect of aPDT with chlorophyllin-phycocyanin mixture (Photoactive+) as PS on E. faecalis and irradiation using different laser wavelengths (635 nm and 660 nm) and light-emitting diode (LED) source (420-480 nm) and the combination of red laser and LED, to improve the efficacy of aPDT.

Bacterial Strain and Culture Conditions
E. faecalis strain ATCC 29,212 obtained from the Iranian Biologic Resource Center (Tehran, Iran) was used in this study. This microorganism was aerobically grown in fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37 • C until logarithmic growth phase to produce a final concentration of 1.5 × 10 8 colony forming unit (CFU)/mL. The concentration of bacteria was approved by spectrophotometry at an optical density (OD) of 600 nm which was 0.08-0.13.

Photosensitizer and Light Sources
Photoactive+ (W Medical system GMBH, Germany) in liposomal form was used as a PS in this study. The peak absorption of this PS was assessed by spectroscopy (Alpha, China). The solution at final concertation of 0.02 g/mL was prepared in distilled water. The solution was kept under dark conditions before use. Diode lasers with wavelengths of 660 nm (DX61, Konftec, Taiwan) with continuous wave (cw) output power of 150 mW, 635 nm (DX62, Konftec, Taiwan) with cw-output power of 220 mW, LED (DY400-4, Denjoy, China) at a wavelength of 450 ± 30 nm with an output intensity of 1000-1400 mW/cm 2 were used ( Figure 1). The output powers of all wavelengths were measured and confirmed using a power meter (Laser Point S.r.l, Milan, Italy).

Antimicrobial Photodynamic Therapy of Bacterial Suspensions
The antimicrobial effect of Photoactive+ against E. faecalis was determined by the broth microdilution method as recommended by the clinical and laboratory standards institute (CLSI) [15]. 150 µL of bacterial suspension at a final concentration of 1.5 × 10 8 CFU/mL was transferred to each well of a 96-well round-bottomed sterile polystyrene microplate (TPP; Trasadingen, Switzerland). The Photoactive+ solution (150 µL) was added to each well. The final concentration of Photoactive+ was 0.01 g/mL. The microplates were incubated for 5 min in the dark at room temperature at 25 ± 2 • C. The wells were then exposed to light sources with different wavelengths and exposure times as listed in Table 1.

Colony Count Assessment
After treatment 10 µL of each well was transferred to the wells that contained 100 µL BHI broth and serial diluted 10-fold stepwise from column one to column five. 10 µL of each dilution was cultured on BHI agar plates (Merck, Darmstadt, Germany) and incubated for 24 h at 37 • C. Microbial viability assay was performed, according to a previously reported method [16]. The number of colony forming units (CFU)/mL was calculated using Miles and Misra method [17].

Cytotoxicity Assay
Primary human gingival fibroblast (HuGu) cell obtained from the Iranian Biologic Resource Center (Tehran, Iran) was cultured in Dulbecco's modified Eagle's medium (DMEM, HiMedia Labs, India) supplemented with 10% fetal bovine serum (FBS; Sigma, USA), L-glutamine (2 mM), 1% penicillin/streptomycin antibiotic solution (10,000 Unit/mL penicillin and 10-mg/mL streptomycin) and 100 g/mL of amphotericin B. After incubation at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 , 5 × 10 3 cells per well were placed in flat-bottom 96-well cell culture microplate and incubated for 24 h at 37 • C in a humidified CO 2 incubator. Subsequently, 100 µL of Photoactive+ at final concentration of 0.01 g/mL were added to wells and the microplate was incubated at 37 • C for 24 h in a humidified atmosphere of 95% air and 5% CO 2 . For measuring cell viability, the MTT (3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-SH-tetrazolium bromide) assay was performed according to published protocol [18] and the absorbance values of each well were read at 570 nm through enzyme-linked immuno-assay (ELISA) plate reader. An inverted microscope (Olympus, India) at magnification of 40× was used to assess the cell morphology before and after treatment.

Statistical Analysis
All experiments were performed in triplicate. Data from the experimental groups were analyzed by using two-way analysis of variance (ANOVA) followed by Tukey's test. The results were presented as mean ± standard deviation. Significance was defined as p-values < 0.05.

Results
The E. faecalis count with aPDT treatments using Photoactive+ activated by red wavelengths (635 nm and 660 nm) and combination of LED (420-480 nm) and red wavelengths (635 nm and 660 nm) were significantly reduced compared to the control group in both irradiation time (p < 0.05) (Figure 1). aPDT with LED (420-480 nm) decreased the bacterial count, but with no significant difference from the control group in both energy density of 26.4 and 76 J/cm 2 (p = 0.75, p = 0.5). The Photoactive+ without activation reduced the count of bacteria with no significant difference from control group. In addition, all light sources including 635 nm, 660 nm and LED (420-480 nm) showed no significant difference in bacteria reduction compared to control group. The greatest reduction in bacterial count was seen with 635-nm red laser activation for energy density of 76 J/cm 2 (83%) and lowest with the Photoactive+ without activation (0.8%). Conversely, considering the energy density, the higher energy density of 76 J/cm 2 showed a higher reduction of the bacterial count compared to lower energy density of 26.4 J/cm 2 in all groups ( Figure 1). The mean and standard deviation of experimental groups were shown in Table 2. The peak absorption of Photoactive+ which assessed by company was shown in Figure 2a (Source: https://www.wmedicalsystems.com/) and our assessment confirmed its absorption in red (620-660 nm) and blue (400-450 nm) region of electromagnetic spectrum (Figure 2b). The results of MTT assay for Photoactive+ at concentration of 0.01-g/mL showed that after the incubation period, there was no significant reduction in the number of viable cells compared to the control group (p > 0.05; Figure 3a). Additionally, morphologically, HuGu of the control group showed a spindle-shaped cell and those in contact with the Photoactive+ demonstrated the typical stellate appearance of this type of cell (Figure 3b,c). The results of MTT assay for Photoactive+ at concentration of 0.01-g/mL showed that after the incubation period, there was no significant reduction in the number of viable cells compared to the control group (p > 0.05; Figure 3a). Additionally, morphologically, HuGu of the control group showed a spindle-shaped cell and those in contact with the Photoactive+ demonstrated the typical stellate appearance of this type of cell (Figure 3b,c).

Discussion
Algae have been used within food and medicine for many centuries. Spirulina-as a blue-green alga-has antioxidant, anticancer, immunomodulation and antimicrobial effects. These effects can be attributed to some components such as phycocyanin [19]. Phycocyanin is a natural blue-colored protein complex that is water-soluble and has a peak absorption between 580-660 nm. This material showed some potential biologic activities that can be considered as a PS when irradiated using the appropriate wavelength [11]. The level of singlet oxygen production is not increased in the non-irradiated phycocyanin group, which confirms that the generation of singlet oxygen is dependent on laser irradiation [10].
Chlorophyll derivates composed of chlorophyllin metal complexes enjoy advantages such as good water-solubility and high photosensitivity and excitation with wavelength in the region of visible red (650-670 nm). This mixture can produce ROS-especially 1O2 in a higher amount and OH° in less amount-when irradiated with lasers of visible red range of wavelengths [20]. The potential of ROS production of this agent from the higher level to lower one is in this order: Fe-CHL > Mg-CHL > Cu-CHL. Hence, Fe-CHL shows the strongest photodynamic action [21]. In this study, we used Photoactive+ as a PS which includes phycocyanin and chlorophyll. Since chlorophyll is not water soluble, it is poorly absorbed by the body. The chlorophyll part in Photoactive+ has changed to possess the ability to dissolve in water in forms of sodium-magnesium-chlorophyllin (200 mg) and sodium-copper-chlorophyllin (100 mg). In addition, this mixture has 200 mg phycocyanin. Phycocyanin is water soluble and can be absorbed by the body. For highest bioavailability, in Photoactive+, the phycocyanin is embedded in a liposome to improve its absorbance in body. Liposomal structure of phycocyanin can incorporate hydrophilic, hydrophobic and amphiphilic substances to achieve improved infiltration of the PS into microorganisms.
We prepared the final concentration of 0.01 g/mL for our experiment. The chlorophyllin can be activated by either red or blue wavelengths or a combination of both. The results of this study showed that aPDT with Photoactive+ and 635 nm diode laser showed the greatest reduction in the

Discussion
Algae have been used within food and medicine for many centuries. Spirulina-as a blue-green alga-has antioxidant, anticancer, immunomodulation and antimicrobial effects. These effects can be attributed to some components such as phycocyanin [19]. Phycocyanin is a natural blue-colored protein complex that is water-soluble and has a peak absorption between 580-660 nm. This material showed some potential biologic activities that can be considered as a PS when irradiated using the appropriate wavelength [11]. The level of singlet oxygen production is not increased in the non-irradiated phycocyanin group, which confirms that the generation of singlet oxygen is dependent on laser irradiation [10].
Chlorophyll derivates composed of chlorophyllin metal complexes enjoy advantages such as good water-solubility and high photosensitivity and excitation with wavelength in the region of visible red (650-670 nm). This mixture can produce ROS-especially 1O2 in a higher amount and OH • in less amount-when irradiated with lasers of visible red range of wavelengths [20]. The potential of ROS production of this agent from the higher level to lower one is in this order: Fe-CHL > Mg-CHL > Cu-CHL. Hence, Fe-CHL shows the strongest photodynamic action [21]. In this study, we used Photoactive+ as a PS which includes phycocyanin and chlorophyll. Since chlorophyll is not water soluble, it is poorly absorbed by the body. The chlorophyll part in Photoactive+ has changed to possess the ability to dissolve in water in forms of sodium-magnesium-chlorophyllin (200 mg) and sodium-copper-chlorophyllin (100 mg). In addition, this mixture has 200 mg phycocyanin. Phycocyanin is water soluble and can be absorbed by the body. For highest bioavailability, in Photoactive+, the phycocyanin is embedded in a liposome to improve its absorbance in body. Liposomal structure of phycocyanin can incorporate hydrophilic, hydrophobic and amphiphilic substances to achieve improved infiltration of the PS into microorganisms.
We prepared the final concentration of 0.01 g/mL for our experiment. The chlorophyllin can be activated by either red or blue wavelengths or a combination of both. The results of this study showed that aPDT with Photoactive+ and 635 nm diode laser showed the greatest reduction in the bacterial count, but when activated by LED (420-480 nm) did not significantly reduce the bacterial count. In addition, our results showed that R1-Blue was better than the Blue-R1 treatment. This can be related to the phenomenon that this complex was more activated by red wavelength rather than blue one. It seemed that that the chlorophyllin part of this mixture had no contribution as an antimicrobial photosensitizer when activated by LED. If a blue laser with a more accurate wavelength were used, it may have had an effect on bacterial reduction.
In agreement with our results, Afrasiabi et al. in assessing the efficacy of chlorophyllin-phycocyanin mixture activated by 635 nm diode laser against Streptococcus mutans cultured on enamel slabs concluded that this mixture can reduce the number of living bacteria within the biofilms of S. mutans [22].
Luksiene et al. evaluated the effect of chlorophyllin-Sodium-Copper activated by blue light (400 nm) with power density of 20 mW/cm2 on Bacillus cereus (Gram-positive bacteria) which led to a significant reduction in bacteria [23]. In addition, Caires et al. used chlorophyllin-Sodium-Copper using a homemade LED device at wavelength of 625 nm and power density of 8.3 mW/cm2 during 1 h for inactivation of Staphylococcus aureus and Escherichia coli. The results of their studies showed efficacy for inactivation of Gram-positive bacteria. The results of our study were in line with above-mentioned studies, as it focused on E. faecalis which is a Gram-positive bacterium [24]. Our result showed that chlorophyllin-phycocyanin mixture at concentration of 1000 µg/mL has a very negligible cytotoxicity (p > 0.05) and can be used safely as a natural compound for aPDT.
Fimple et al. in assessing the PDT for eradication of polymicrobial infection in endodontic concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized. On the other hand, due to minimally invasive manner of this technique it can be beneficial to be used as an adjunct treatment [25,26].
This study carried out using a standard strain of E. faecalis in in vitro condition which can be considered as a limitation of the study. Anatomic complexity of root structure and morphology, oxygen presence, heterogeneity of clinical isolates of E. faecalis with different behavior and resistance properties can be the reasons which may limit direct extrapolation of this study to embrace clinical conditions. Nonetheless, the irradiation timing values used would find clinical acceptance and it may be possible to extend laser irradiation to assess the degree antimicrobial efficacy. However, the use of aPDT, based on Photoactive+ as adjunctive to conventional chemo-mechanical debridement of infected root canal system provides additional benefits. Further high-quality randomized clinical trials focused on the standardized aPDT parameters and high methodological quality are needed.

Conclusions
The results of this study demonstrate that the efficacy of aPDT by Photoactive+ is dependent on the light source wavelength with the superiority of red laser compared to the blue light source. It is recommended to use the Photoactive+ as a PS accompanied by red diode lasers or combination of red diode laser first following the blue wavelength. The chlorophyllin-phycocyanin mixture can be applied safely due to very negligible cytotoxicity.