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Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation
Journal Pre-proof Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation Jennifer Machado Soares, Karoliny Oliveira Ozias Silva, Natalia Mayumi Inada, Vanderlei Salvador Bagnato, Kate Cristina Blanco
PII:
S1572-1000(20)30004-1
DOI:
https://doi.org/10.1016/j.pdpdt.2020.101652
Reference:
PDPDT 101652
To appear in:
Photodiagnosis and Photodynamic Therapy
Received Date:
28 September 2019
Revised Date:
19 December 2019
Accepted Date:
3 January 2020
Please cite this article as: Soares JM, Silva KOO, Inada NM, Bagnato VS, Blanco KC, Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation, Photodiagnosis and Photodynamic Therapy (2020), doi: https://doi.org/10.1016/j.pdpdt.2020.101652
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation
Jennifer Machado Soares*, Karoliny Oliveira Ozias Silva, Natalia Mayumi Inada, Vanderlei Salvador Bagnato, and Kate Cristina Blanco,
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São Carlos Institute of Physics, University of São Paulo – Box 369, 13566-970, São Carlos, SP,
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Brazil
*Corresponding author: Av. Trabalhador São-carlense, 400, São Carlos, São Paulo, Brazil,
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Postal code: 13566-590, +55 (16) 3373-9810, Fax: 3373-9811. Email: [email protected]
Highlights
Absorption of curcumin by bacteria is favored by the formulation
The presence of carbohydrate exerts an influence on the PS interaction with bacteria.
The interaction between curcuminoids may favor their solubilization and affect the interaction with the bacterial cell
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Abstract
INTRODUCTION: A mixture of curcuminoids: curcumin, desmethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are named natural curcumin. It is a lipophilic photosensitizer (PS) highly soluble in an organic solvent such as dimethyl sulfoxide (DMSO). Curcumin is a PS used for microbial inactivation using photodynamical action. However, this
solvent has high cytotoxicity and unavailable in formulations for clinical use. This study aimed to investigate the interactions of curcuminoids syrup with Streptococcus sp., a grampositive coccus and one of the major pharyngeal pathogens, responsible for diseases such as pharyngitis. METHODS: bacteria were incubated with curcuminoids (natural curcumin, synthetic, DMC, BDMC) at 37 ° C in formulations: 1) syrup (water + sucrose) 2) solution alcohol + DMSO. Was centrifuged, and the supernatant collected for absorbance analysis. The results obtained correlating the absorbance with the supernatant to the absorbance of the
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default concentration. A study of microbial metabolism by growth curve was carried out to justify the result. RESULTS: The incorporation of curcumin in syrup is superior to
alcohol/DMSO solution by microorganisms. Curcumin incorporation by S. mutans, S.
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pyogenes, isolated bacteria was 24, 26, 27% in syrup and 10, 13, 5% in alcohol/DMSO, respectively. Also, the presence of carbohydrate in a solution can activate the bacterial
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metabolism, getting better uptake results and photodynamic inactivation to natural curcumin
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and DMC. Such finds care optimizes the use of curcumin without complications generated by
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the solvent.
Keywords: Photosensitizer; Curcumin; Streptococcus sp.; Pharyngitis; Photodynamic
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Therapy
1. Introduction
Even considering that the palatine tonsils contain microbial biota, including anaerobic
and aerobic bacteria, several etiologic agents can cause pharyngotonsillitis in both adults and children. The leading causes of bacterial pharyngotonsillitis (PT) are Streptococcus pyogenes, a Beta-hemolytic Group A Streptococcus (BGAS). In general, the incidence of each
microorganism depends on the age of the patient, geographic region, and time of year considered. The main etiologic agents and incidence of acute pharyngotonsillitis caused by BGAS are up to 21,9% in adults (MCISAAC et al., 2004) and up to 50% in children (MAZUR et al., 2011). Other streptococci B hemolytic with a frequency of 1% -9.5% can be the cause of PT (BA-SADDIK et al., 2014; MAZUR et al., 2011). The conventional treatment of PT consists of the administration of antibiotics such as penicillin. The PT may evolve rheumatic fever, which can lead to death (EFSTRATIOU; LAMAGNI, 2016). Photodynamic
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Therapy (PDT) is an alternative to standard treatment for cancer and infections. The mechanism of PDT allows treatments to be localized so that only the microorganisms of the
infection are eliminated, thus maintaining the commensal bacteria of the human body. It is a
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treatment that does not promote the selection of resistant microorganisms, since different
from antibiotic, the PDT does not have a single cellular or molecular target for its action. The
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PDT action principle involves a PS molecule that absorbs the light in a specific region of the
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electromagnetic spectrum. The absorption of light by PS enables the change to the excited state, which triggers the production of reactive species in two ways (ABRAHAMSE; HAMBLIN, 2016). The first type of reaction is related to the transfer of an electron to the
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biomolecules present in the cells, consequently, form reactive oxygen species (ROS). The second type transfers energy to molecular oxygen, which becomes to the excited singlet state.
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These ROS can react with protein, lipids, and nucleic acids, promoting cell death
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(ROBERTSON et al., 2009). The effectiveness of the PDT depends on the type of PS and light intensity and fluency needed to promote the microbial reduction. The PS and the light alone do not cause any action. Natural curcumin is a PS composed of curcumin (CUR), desmethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), which are obtained from the alcoholic extraction of Curcuma longa rhizome (SUETH-SANTIAGO et al., 2015). Across the use nutritional and as
PS, the natural curcumin as bioactive proprieties with anti-inflammatory, antiangiogenic, antioxidant, healing, and anticancer effects. Due to its low toxicity and few sides effects, it is considered safe by regulatory agencies such as the Food and Drug Administration (FDA) of the United States of America (FDA ; DOLCAS BIOTECH, 2016). The structure of curcumin [(1E,6E) -1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-heptadiene3,5- dione] is composed of two aromatic units substituted by a methoxy group and a phenolic group, both separated by polyethylene chain composed of keto-enol or di-keto group. The
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curcumin is found predominantly in the enol form when diluted in ethanol, but in an aqueous solvent, the di-keto form is predominant stabilized by the coordination of a water molecule (BERNABÉ-PINEDA et al., 2004). The structural characteristics confer the curcumin
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lipophilic character (Log P = 2.5) an acid, highly soluble in an organic solvent such as
dimethyl sulfoxide (DMSO), methanol, ethanol, and acetone (COOKSEY, 2017). However,
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these solvents have high toxicity in cells which unavailable use for clinical formulation.
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Sucrose in water may aid the solubilization and stability of curcuminoids, allowing the use of a formulation likely to be used in clinical applications without risk of solvent toxicity.
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For the PDT effectiveness, it is pivotal that the ROS produced in the process interact with the microorganisms and its cellular structures, leading them to the destruction. This interaction
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may be more efficient and quantified by the incorporation of a molecule by the bacteria, which may be different according to the proprieties of medium and solvent present used in the
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PDT procedure.
In this work, we investigated the interactions of curcumin in two different formulations: a
syrup containing water and sugar, and a standard formulation of inorganic solvents (ethyl alcohol and DMSO). For this, natural curcumin extracted from the root of Curcuma longa, DMC, BDMC and synthetically obtained curcumin were used.
2. Materials and methods 2.1 Preparation of microorganism Streptococcus mutans (ATCC 25923), Streptococcus pyogenes, and clinical isolates both from patients diagnosed with PT were cultivated in Brain Heart Infusion (BHI) at 37 ◦C and 150 rpm overnight. The samples were centrifuged at 3000 rpm for 10 min and resuspended in phosphate-buffered saline (PBS) and subsequently diluted for obtaining of bacterial inoculum
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of 108 CFU/ml (colony forming units per milliliters) by optical density at 600 nm (Cary UVVis50, Varian). 2.2 Preparation of photosensitizer
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2.2.1 Curcumin in alcohol and DMSO
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To prepare a stock solution, dissolved 5 mg of natural curcumin (PDT PHARMA®) in
2.2.2 Curcuminoids syrup
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dimethylsulfoxide DMSO 10 µL and 1 mL of ethyl alcohol in 0.5; 0.75; 1; 3 and 5 mg/ml.
The syrup base was prepared with 30% sucrose at 80° C on the thermal plate. The stock
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solution of syrup as prepared using 5 mg of natural curcumin, synthetic curcumin, bisdemethoxycurcumin, and desmethoxycurcumin (PDT PHARMA®) macerated and added
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into syrup base at ethyl alcohol 2% (w/v) (BLANCO et al., 2017). Concentrations of 0.5;
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0.75; 1; 2.25; 3 and 5 mg/ml of PS were obtained from the stock solution. Curcumin solutions prepared at a concentration of 2.25 mg/ml. 2.3 Quantification of interaction of photosensitizer The bacterial inoculum of 108 CFU/ml centrifuged for 10 minutes at 3000 rpm. The supernatant discarded, and the pellet resuspended in a solution containing PS and incubated at 37 ° C for 4 minutes in the dark. After this time, the samples centrifuged at 3300 rpm for 3
minutes, and the supernatant collected. Five dilutions were performed to measure the absorbance in Spectrophotometer UV-vis (Cary UV-Vis50, Varian) at 425 ± 7 nm for curcumin in DMSO and at 440 ± 7 nm for the curcumin in syrup. The results obtained across the correlation between the absorbance of supernatant and absorption of standard concentration (GEORGE et al., 2009). The percentage of uptake was calculated using the formula below. 𝐴𝑏𝑠𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 ) 𝑥100 𝐴𝑏𝑠𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
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𝑈𝑝𝑡𝑎𝑘𝑒(%) = (1 −
2.4 Antimicrobial Photodynamic Therapy
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2.4.1 Light source
The device used for the irradiation of the samples was a Biotable® developed by the
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Laboratory of Technical Support of the São Carlos Institute of Physics. It is composed of 24 wells containing LED in the wavelength of 450 nm. Each well receives uniform irradiation
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with an average intensity of 40 mW/cm2 and a total fluency of 28,8 and 60 J/cm2.
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2.4.2 Inactivation photodynamic
The bacteria (108 CFU/ml) resuspended in PBS submitted to PDT with the Biotable®
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device. Each experiment has two groups, with three repetitions each: 1) only bacteria, bacteria with PS and bacteria irradiated; 2) bacteria with PS irradiated. All samples were
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diluted and plated in Petri dishes with BHI agar medium and incubated at 37 ° C for 24 hours.
2.5 Growth curve metabolism analysis After microorganism preparation (session 2.1), the bacterial inoculum resuspended in distilled water and syrup base at the ratio of 1:400, and then placed in the shaker at 150 rpm at 37 ° C.
Optical density measurements were performed at 600 nm by spectrophotometer (Cary UVVis50, Varian). 2.6 Statics analyses All experiments performed in triplicate of three different events (total n=9). The standard deviation shown error bars in some cases, due to the small value, the bar is not noticeable. The Student's t-test evaluated the results. P. value <0.05; 0.01; 0.005; 0.001 was considered
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significant. Results
Curcumin has maximum absorption at λ = 440 nm and λ = 425 nm in syrup and
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alcohol / DMSO, respectively. Quantification of curcumin uptake performed on the
incubation period of the microorganism. The correlation is the absorbance of the supernatant
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and the nominal concentration of the formulation for 4 minutes. The interaction of syrup
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curcumin compared with alcohol/DMSO solution to different bacterial strains: S. mutans (ATCC 25923), an oral pathogen; S. pyogenes, a clinical isolate of pharyngotonsillitis; and
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clinical isolates from patients with pharyngotonsillitis. Figure 1 (A, C, E) shows that the formulation favors the absorption of curcumin by bacteria, and syrup promoted greater
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incorporation of PS when compared to alcohol / DMSO. The average percentage uptake of curcumin by S. mutans, S. pyogenes, and clinical isolated was 24, 26, and 27% in syrup and
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10, 13, and 5% in alcohol/DMSO solution, respectively (Figure 1-A, C, E). The syrup interaction is predominant at low concentrations by clinical isolated as shown in
Figure 1, panel C. S. mutans and S. pyogenes strain presented linear uptake rate at the concentration available up to the threshold of 3 mg/ml (Figure 1-A, C). The concentrations of PSs may interfere with the mechanism of saturation of their penetration by bacteria in which values above 3 mg/ml tends to decrease their uptake in three microbial strains slightly.
Antimicrobial photodynamic therapy (aPDT) was performed after PS incorporation by microorganisms, considering the light dose of 28.8J/cm² at 450 nm for the different concentrations of syrup. To show the results, the analysis of CFU/ml data were transformed to logarithmic base (log 10), as shown in Figure 1- B, D, F. A reduction of 3.4 log of S. mutans (panel B), 2.9 logs of S. pyogenes (panel D) and 2.5 of isolated bacteria from PT (panel F) at the nominal concentration of 5 mg/ml obtained. These reductions are approximately similar to the nominal concentration of 3mg/ml. The interaction of the separate components, only light
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or curcumin syrup, showed no cytotoxic effects (data not shown). Inactivation of clinical isolates is lower than in isolated strains. Therefore, the antimicrobial activity of curcumin in alcohol / DMSO against bacterial strains not presented due to its toxicity in the absence of
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light.
The microbial growth curve investigated the influence of syrup on bacterial metabolism, and
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differences in PS incorporation in different formulations were observed (Figure 2). The
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strains show a negative concavity growth pattern for syrup. However, the concavity becomes positive with no sucrose, which linked to the cell growth rate. The microbial growth in syrup
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is accelerated in the first hours (Figure 2-A, B), rapidly reaching a stationary phase when compared to the growth in distilled water without sucrose. The presence of nutrients for
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microbial metabolism occurs necessarily. The presence of sucrose allows for statistically significant growth in the first few hours, as shown in Figure 2-A, B while in Figure 2-C, the
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growth differentiation becomes significant 5 hours after, probably due to the clinical isolate be more nutritionally demanding. Therefore, the results are suggesting that the presence of carbohydrate exerts an influence on the PS interaction with bacteria. Since there was a better interaction of natural curcumin in the syrup formulation, it was investigated the improvement of aPDT using different incubation times for different curcuminoids. Figure 3 shows the results of the interaction of syrup with S. pyogenes (panel
A) and aPDT (panel B), for different curcuminoids using the concentration below the stagnation threshold of 3 mg/ml. Overall, the best incubation time presented for the different syrup curcuminoids was at 12 minutes, as it reached the highest uptake value. Homogeneous emulsion of natural curcumin and DMC resulted in an inactivation of 4.2 log (CFU / ml) and 3.1 log (CFU/ml), respectively for S. pyogenes. An inhomogeneous emulsion with synthetic curcumin and BDMC resulted in a lower bacterial inactivation of 1.5 log (CFU / ml) and 2.9 log (CFU / ml), respectively.
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4. Discussion
Antimicrobial Resistance has increased significantly over the years, with an estimated 10 million deaths a year globally in 2050 (O’NEILL J., 2016). PDT for the treatment of PT
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based on in vitro studies has been previously proven using Staphylococcus aureus as a
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promising technique for clinical use (BLANCO et al., 2017).
The results suggest that syrup formulation may directly influence the interaction rate of PS
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with the microorganism. Similarly, it verified by BHAWANA et al., 2011 that the photoantimicrobial activity of the nanoparticle of curcumin in water was higher than the activity of
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curcumin in DMSO. And SHLAR et al., 2017 showed that curcumin's antimicrobial action depends on the administration system. The nutritional requirement for a carbon source by the
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bacterial strains observed by the replication time observed in the growth curve of the microorganisms. This result suggests that the absorption of curcuminoids by bacteria
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increases with the rate of microbial metabolism, considering that strains of the genus Streptococcus of clinical relevance are heterotrophic bacteria capable of metabolizing glucose (WINN; KONEMAN, 2006). Therefore, the presence of carbohydrates in an environment accelerates the metabolism of microorganisms and, consequently, the uptake of sucrose molecules that carry curcumin molecules and other curcuminoids. This mechanism is similar to the strategy used to administer antibiotics in a resistant microorganism to drugs (ALLISON
et al., 2011). In addition to sugar, other components may assist solubilize curcuminoids and affect cell uptake, such as bovine serum albumin (ITAYA et al., 2019). Whereas the susceptibility of bacteria to antibiotics is related to their metabolism, bacterial strains susceptible to antibiotics have a higher metabolic rate than antibiotic-resistant strains. The drug administered with a compound to metabolized may facilitate the absorption of the therapeutic compound. Glucose and fructose can increase the NADH production and protonmotor force due to the activation of the citric acid cycle (CAC). Thus, the use of a metabolite
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stimulates the incorporation of a drug, especially for resistant strains that have a slower metabolism (PENG et al., 2015).
The biological membrane is the main cellular component of interaction with amphipathic
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molecules such as curcumin, which has the main hydrophobic chain with some polar
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functional groups that are capable of interacting with the biological membrane and influences the fluidity similar to other amphipathic molecules such as cholesterol (SUN et al., 2008). The
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head of the phospholipids interacts with the methoxy or hydroxyl groups of curcumin — the direction of binding influences differently on the degree of order of the bilayer. The
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interaction of curcumin at low concentrations occurs only on the surface of the membrane with the ordered acyl groups; however, when the connection is perpendicular are disordered.
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Curcumin can present a steric hindrance in the binding region by promoting arrangements locally since the non-binding region can exhibit a disorder for entropic compensation
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(BARRY et al., 2009). This modulation of the membrane is similar to the mechanism of eukaryotic cells with the presence of cholesterol in the membrane; however, the bacterial mechanical stability is maintained by a cell wall, since cholesterol is absent. However, the effects of curcumin in the biologic membrane are not all understood yet. In addition to interaction with the phospholipids, curcumin can modulate the activity of membrane proteins as verified by INGOLFSSON et al., 2007 and HUNG et al., 2008 by studying the channels
formed by gramicidin A without affecting its conductance that modify the thickness and elastic property of the bilayers. Membrane integrity studies were performed with propidium iodide and calcein and verified that the protein has membrane damaging property (TYAGI et al., 2015). Bacterial cell death caused by PDT results from damage to the cytoplasmic membrane and DNA. Membrane proteins are modified by damage which alter concentration gradients and bacterial cell wall synthesis, factors that are sufficient to induce microbial death. The quantum
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efficiency may differ depending on location them hiding the diffusion of ROS in molecular targets (HAMBLIN; HASAN, 2004). However, internalization making the process more effective; it allows the action of the two pathways: type I (electron transfer) requires
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internalization; type II (energy transfer) is not necessary. Thus, the incubation times allow the localization of PS in the cell, which will aid in the diffusion of the reactive species and
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consequently the efficiency of the photodynamic action, especially for the type I reactions that
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should preferably occur in the cellular interior for greater efficacy, because there are larger biomolecules available for iteration (BACELLAR et al., 2015).
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There are several studies with different compounds of the pharmaceutical form which aim to aid the solubility of curcuminoids in water for clinical applications. Our results suggest that
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the interaction between curcuminoids may favor their solubilization and affect the interaction with the bacterial cell. In addition to PS uptake, it is important that the formulation is
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homogeneous to promote photodynamic inactivation. In the case of natural curcumin and DMC were the molecules with better solubility and homogeneity of the formulation compared to the synthetic and BDMC. Besides that, a bacterial community of multiple species has symbiosis relationships against external agents that explains the lower efficiency of aPDT. Appropriate curcumin uptake and understanding of the model presented here to assist the legitimate use of syrup in clinical application.
5. Conclusion The uptake of curcumin by bacteria is an essential step to an efficient aPDT. Besides, the physicochemical characteristics of the molecule and the uptake rate can be modulated by the pharmaceutical formulations, allowing an alternative strategy to implementation of treatments in clinical cases when the PS is more soluble in cytotoxic. Our results are suggesting that the use of curcuminoids in syrup favors the incorporation of this PS, mainly natural curcumin and DMC, improving the clinical protocols for antimicrobial control,
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allowing the use of lipophilic molecules as curcumin in aqueous solutions. Acknowledgement
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
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Superior - Brasil (CAPES) - Finance Code 001 and Center of Optics and Photonics (CEPOF-
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Fapesp 2013/07276-1). As well as CNPQ INCT program.
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Figures
Figure 1: Uptake of different concentrations of curcumin syrup, curcumin in alcohol/DMSO
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and aPDT for different concentrations of curcumin syrup incorporated into bacteria. A) uptake and B) aPDT of S. mutans. C) Uptake and D) aPDT of S. pyogenes. E) uptake and D) aPDT
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of clinical isolated from PT. *; **; ***, # indicates a statistical significance of p<0.05; 0.01;
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0.005 and 0.001, respectively.
Figure 2: Microbial grown curve (600 nm) with or without sucrose (syrup): A) S. mutans.
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B) S. pyogenes; C) clinical isolated of PT.
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Figure 3: Interaction of different curcuminoids (natural, synthetic, desmethoxycurcumin and bisdemethoxycurcumin) in syrup with S. pyogenes in 2.25 mg/ml. A) Rate of uptake.
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B) Reduction of log (CFU/ml) by aPDT with 60 J/cm².
PII:
S1572-1000(20)30004-1
DOI:
https://doi.org/10.1016/j.pdpdt.2020.101652
Reference:
PDPDT 101652
To appear in:
Photodiagnosis and Photodynamic Therapy
Received Date:
28 September 2019
Revised Date:
19 December 2019
Accepted Date:
3 January 2020
Please cite this article as: Soares JM, Silva KOO, Inada NM, Bagnato VS, Blanco KC, Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation, Photodiagnosis and Photodynamic Therapy (2020), doi: https://doi.org/10.1016/j.pdpdt.2020.101652
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation
Jennifer Machado Soares*, Karoliny Oliveira Ozias Silva, Natalia Mayumi Inada, Vanderlei Salvador Bagnato, and Kate Cristina Blanco,
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São Carlos Institute of Physics, University of São Paulo – Box 369, 13566-970, São Carlos, SP,
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Brazil
*Corresponding author: Av. Trabalhador São-carlense, 400, São Carlos, São Paulo, Brazil,
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Postal code: 13566-590, +55 (16) 3373-9810, Fax: 3373-9811. Email: [email protected]
Highlights
Absorption of curcumin by bacteria is favored by the formulation
The presence of carbohydrate exerts an influence on the PS interaction with bacteria.
The interaction between curcuminoids may favor their solubilization and affect the interaction with the bacterial cell
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ur
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Abstract
INTRODUCTION: A mixture of curcuminoids: curcumin, desmethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are named natural curcumin. It is a lipophilic photosensitizer (PS) highly soluble in an organic solvent such as dimethyl sulfoxide (DMSO). Curcumin is a PS used for microbial inactivation using photodynamical action. However, this
solvent has high cytotoxicity and unavailable in formulations for clinical use. This study aimed to investigate the interactions of curcuminoids syrup with Streptococcus sp., a grampositive coccus and one of the major pharyngeal pathogens, responsible for diseases such as pharyngitis. METHODS: bacteria were incubated with curcuminoids (natural curcumin, synthetic, DMC, BDMC) at 37 ° C in formulations: 1) syrup (water + sucrose) 2) solution alcohol + DMSO. Was centrifuged, and the supernatant collected for absorbance analysis. The results obtained correlating the absorbance with the supernatant to the absorbance of the
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default concentration. A study of microbial metabolism by growth curve was carried out to justify the result. RESULTS: The incorporation of curcumin in syrup is superior to
alcohol/DMSO solution by microorganisms. Curcumin incorporation by S. mutans, S.
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pyogenes, isolated bacteria was 24, 26, 27% in syrup and 10, 13, 5% in alcohol/DMSO, respectively. Also, the presence of carbohydrate in a solution can activate the bacterial
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metabolism, getting better uptake results and photodynamic inactivation to natural curcumin
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and DMC. Such finds care optimizes the use of curcumin without complications generated by
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the solvent.
Keywords: Photosensitizer; Curcumin; Streptococcus sp.; Pharyngitis; Photodynamic
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Therapy
1. Introduction
Even considering that the palatine tonsils contain microbial biota, including anaerobic
and aerobic bacteria, several etiologic agents can cause pharyngotonsillitis in both adults and children. The leading causes of bacterial pharyngotonsillitis (PT) are Streptococcus pyogenes, a Beta-hemolytic Group A Streptococcus (BGAS). In general, the incidence of each
microorganism depends on the age of the patient, geographic region, and time of year considered. The main etiologic agents and incidence of acute pharyngotonsillitis caused by BGAS are up to 21,9% in adults (MCISAAC et al., 2004) and up to 50% in children (MAZUR et al., 2011). Other streptococci B hemolytic with a frequency of 1% -9.5% can be the cause of PT (BA-SADDIK et al., 2014; MAZUR et al., 2011). The conventional treatment of PT consists of the administration of antibiotics such as penicillin. The PT may evolve rheumatic fever, which can lead to death (EFSTRATIOU; LAMAGNI, 2016). Photodynamic
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Therapy (PDT) is an alternative to standard treatment for cancer and infections. The mechanism of PDT allows treatments to be localized so that only the microorganisms of the
infection are eliminated, thus maintaining the commensal bacteria of the human body. It is a
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treatment that does not promote the selection of resistant microorganisms, since different
from antibiotic, the PDT does not have a single cellular or molecular target for its action. The
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PDT action principle involves a PS molecule that absorbs the light in a specific region of the
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electromagnetic spectrum. The absorption of light by PS enables the change to the excited state, which triggers the production of reactive species in two ways (ABRAHAMSE; HAMBLIN, 2016). The first type of reaction is related to the transfer of an electron to the
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biomolecules present in the cells, consequently, form reactive oxygen species (ROS). The second type transfers energy to molecular oxygen, which becomes to the excited singlet state.
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These ROS can react with protein, lipids, and nucleic acids, promoting cell death
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(ROBERTSON et al., 2009). The effectiveness of the PDT depends on the type of PS and light intensity and fluency needed to promote the microbial reduction. The PS and the light alone do not cause any action. Natural curcumin is a PS composed of curcumin (CUR), desmethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), which are obtained from the alcoholic extraction of Curcuma longa rhizome (SUETH-SANTIAGO et al., 2015). Across the use nutritional and as
PS, the natural curcumin as bioactive proprieties with anti-inflammatory, antiangiogenic, antioxidant, healing, and anticancer effects. Due to its low toxicity and few sides effects, it is considered safe by regulatory agencies such as the Food and Drug Administration (FDA) of the United States of America (FDA ; DOLCAS BIOTECH, 2016). The structure of curcumin [(1E,6E) -1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-heptadiene3,5- dione] is composed of two aromatic units substituted by a methoxy group and a phenolic group, both separated by polyethylene chain composed of keto-enol or di-keto group. The
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curcumin is found predominantly in the enol form when diluted in ethanol, but in an aqueous solvent, the di-keto form is predominant stabilized by the coordination of a water molecule (BERNABÉ-PINEDA et al., 2004). The structural characteristics confer the curcumin
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lipophilic character (Log P = 2.5) an acid, highly soluble in an organic solvent such as
dimethyl sulfoxide (DMSO), methanol, ethanol, and acetone (COOKSEY, 2017). However,
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these solvents have high toxicity in cells which unavailable use for clinical formulation.
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Sucrose in water may aid the solubilization and stability of curcuminoids, allowing the use of a formulation likely to be used in clinical applications without risk of solvent toxicity.
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For the PDT effectiveness, it is pivotal that the ROS produced in the process interact with the microorganisms and its cellular structures, leading them to the destruction. This interaction
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may be more efficient and quantified by the incorporation of a molecule by the bacteria, which may be different according to the proprieties of medium and solvent present used in the
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PDT procedure.
In this work, we investigated the interactions of curcumin in two different formulations: a
syrup containing water and sugar, and a standard formulation of inorganic solvents (ethyl alcohol and DMSO). For this, natural curcumin extracted from the root of Curcuma longa, DMC, BDMC and synthetically obtained curcumin were used.
2. Materials and methods 2.1 Preparation of microorganism Streptococcus mutans (ATCC 25923), Streptococcus pyogenes, and clinical isolates both from patients diagnosed with PT were cultivated in Brain Heart Infusion (BHI) at 37 ◦C and 150 rpm overnight. The samples were centrifuged at 3000 rpm for 10 min and resuspended in phosphate-buffered saline (PBS) and subsequently diluted for obtaining of bacterial inoculum
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of 108 CFU/ml (colony forming units per milliliters) by optical density at 600 nm (Cary UVVis50, Varian). 2.2 Preparation of photosensitizer
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2.2.1 Curcumin in alcohol and DMSO
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To prepare a stock solution, dissolved 5 mg of natural curcumin (PDT PHARMA®) in
2.2.2 Curcuminoids syrup
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dimethylsulfoxide DMSO 10 µL and 1 mL of ethyl alcohol in 0.5; 0.75; 1; 3 and 5 mg/ml.
The syrup base was prepared with 30% sucrose at 80° C on the thermal plate. The stock
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solution of syrup as prepared using 5 mg of natural curcumin, synthetic curcumin, bisdemethoxycurcumin, and desmethoxycurcumin (PDT PHARMA®) macerated and added
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into syrup base at ethyl alcohol 2% (w/v) (BLANCO et al., 2017). Concentrations of 0.5;
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0.75; 1; 2.25; 3 and 5 mg/ml of PS were obtained from the stock solution. Curcumin solutions prepared at a concentration of 2.25 mg/ml. 2.3 Quantification of interaction of photosensitizer The bacterial inoculum of 108 CFU/ml centrifuged for 10 minutes at 3000 rpm. The supernatant discarded, and the pellet resuspended in a solution containing PS and incubated at 37 ° C for 4 minutes in the dark. After this time, the samples centrifuged at 3300 rpm for 3
minutes, and the supernatant collected. Five dilutions were performed to measure the absorbance in Spectrophotometer UV-vis (Cary UV-Vis50, Varian) at 425 ± 7 nm for curcumin in DMSO and at 440 ± 7 nm for the curcumin in syrup. The results obtained across the correlation between the absorbance of supernatant and absorption of standard concentration (GEORGE et al., 2009). The percentage of uptake was calculated using the formula below. 𝐴𝑏𝑠𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 ) 𝑥100 𝐴𝑏𝑠𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
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𝑈𝑝𝑡𝑎𝑘𝑒(%) = (1 −
2.4 Antimicrobial Photodynamic Therapy
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2.4.1 Light source
The device used for the irradiation of the samples was a Biotable® developed by the
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Laboratory of Technical Support of the São Carlos Institute of Physics. It is composed of 24 wells containing LED in the wavelength of 450 nm. Each well receives uniform irradiation
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with an average intensity of 40 mW/cm2 and a total fluency of 28,8 and 60 J/cm2.
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2.4.2 Inactivation photodynamic
The bacteria (108 CFU/ml) resuspended in PBS submitted to PDT with the Biotable®
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device. Each experiment has two groups, with three repetitions each: 1) only bacteria, bacteria with PS and bacteria irradiated; 2) bacteria with PS irradiated. All samples were
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diluted and plated in Petri dishes with BHI agar medium and incubated at 37 ° C for 24 hours.
2.5 Growth curve metabolism analysis After microorganism preparation (session 2.1), the bacterial inoculum resuspended in distilled water and syrup base at the ratio of 1:400, and then placed in the shaker at 150 rpm at 37 ° C.
Optical density measurements were performed at 600 nm by spectrophotometer (Cary UVVis50, Varian). 2.6 Statics analyses All experiments performed in triplicate of three different events (total n=9). The standard deviation shown error bars in some cases, due to the small value, the bar is not noticeable. The Student's t-test evaluated the results. P. value <0.05; 0.01; 0.005; 0.001 was considered
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significant. Results
Curcumin has maximum absorption at λ = 440 nm and λ = 425 nm in syrup and
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alcohol / DMSO, respectively. Quantification of curcumin uptake performed on the
incubation period of the microorganism. The correlation is the absorbance of the supernatant
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and the nominal concentration of the formulation for 4 minutes. The interaction of syrup
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curcumin compared with alcohol/DMSO solution to different bacterial strains: S. mutans (ATCC 25923), an oral pathogen; S. pyogenes, a clinical isolate of pharyngotonsillitis; and
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clinical isolates from patients with pharyngotonsillitis. Figure 1 (A, C, E) shows that the formulation favors the absorption of curcumin by bacteria, and syrup promoted greater
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incorporation of PS when compared to alcohol / DMSO. The average percentage uptake of curcumin by S. mutans, S. pyogenes, and clinical isolated was 24, 26, and 27% in syrup and
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10, 13, and 5% in alcohol/DMSO solution, respectively (Figure 1-A, C, E). The syrup interaction is predominant at low concentrations by clinical isolated as shown in
Figure 1, panel C. S. mutans and S. pyogenes strain presented linear uptake rate at the concentration available up to the threshold of 3 mg/ml (Figure 1-A, C). The concentrations of PSs may interfere with the mechanism of saturation of their penetration by bacteria in which values above 3 mg/ml tends to decrease their uptake in three microbial strains slightly.
Antimicrobial photodynamic therapy (aPDT) was performed after PS incorporation by microorganisms, considering the light dose of 28.8J/cm² at 450 nm for the different concentrations of syrup. To show the results, the analysis of CFU/ml data were transformed to logarithmic base (log 10), as shown in Figure 1- B, D, F. A reduction of 3.4 log of S. mutans (panel B), 2.9 logs of S. pyogenes (panel D) and 2.5 of isolated bacteria from PT (panel F) at the nominal concentration of 5 mg/ml obtained. These reductions are approximately similar to the nominal concentration of 3mg/ml. The interaction of the separate components, only light
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or curcumin syrup, showed no cytotoxic effects (data not shown). Inactivation of clinical isolates is lower than in isolated strains. Therefore, the antimicrobial activity of curcumin in alcohol / DMSO against bacterial strains not presented due to its toxicity in the absence of
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light.
The microbial growth curve investigated the influence of syrup on bacterial metabolism, and
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differences in PS incorporation in different formulations were observed (Figure 2). The
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strains show a negative concavity growth pattern for syrup. However, the concavity becomes positive with no sucrose, which linked to the cell growth rate. The microbial growth in syrup
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is accelerated in the first hours (Figure 2-A, B), rapidly reaching a stationary phase when compared to the growth in distilled water without sucrose. The presence of nutrients for
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microbial metabolism occurs necessarily. The presence of sucrose allows for statistically significant growth in the first few hours, as shown in Figure 2-A, B while in Figure 2-C, the
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growth differentiation becomes significant 5 hours after, probably due to the clinical isolate be more nutritionally demanding. Therefore, the results are suggesting that the presence of carbohydrate exerts an influence on the PS interaction with bacteria. Since there was a better interaction of natural curcumin in the syrup formulation, it was investigated the improvement of aPDT using different incubation times for different curcuminoids. Figure 3 shows the results of the interaction of syrup with S. pyogenes (panel
A) and aPDT (panel B), for different curcuminoids using the concentration below the stagnation threshold of 3 mg/ml. Overall, the best incubation time presented for the different syrup curcuminoids was at 12 minutes, as it reached the highest uptake value. Homogeneous emulsion of natural curcumin and DMC resulted in an inactivation of 4.2 log (CFU / ml) and 3.1 log (CFU/ml), respectively for S. pyogenes. An inhomogeneous emulsion with synthetic curcumin and BDMC resulted in a lower bacterial inactivation of 1.5 log (CFU / ml) and 2.9 log (CFU / ml), respectively.
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4. Discussion
Antimicrobial Resistance has increased significantly over the years, with an estimated 10 million deaths a year globally in 2050 (O’NEILL J., 2016). PDT for the treatment of PT
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based on in vitro studies has been previously proven using Staphylococcus aureus as a
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promising technique for clinical use (BLANCO et al., 2017).
The results suggest that syrup formulation may directly influence the interaction rate of PS
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with the microorganism. Similarly, it verified by BHAWANA et al., 2011 that the photoantimicrobial activity of the nanoparticle of curcumin in water was higher than the activity of
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curcumin in DMSO. And SHLAR et al., 2017 showed that curcumin's antimicrobial action depends on the administration system. The nutritional requirement for a carbon source by the
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bacterial strains observed by the replication time observed in the growth curve of the microorganisms. This result suggests that the absorption of curcuminoids by bacteria
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increases with the rate of microbial metabolism, considering that strains of the genus Streptococcus of clinical relevance are heterotrophic bacteria capable of metabolizing glucose (WINN; KONEMAN, 2006). Therefore, the presence of carbohydrates in an environment accelerates the metabolism of microorganisms and, consequently, the uptake of sucrose molecules that carry curcumin molecules and other curcuminoids. This mechanism is similar to the strategy used to administer antibiotics in a resistant microorganism to drugs (ALLISON
et al., 2011). In addition to sugar, other components may assist solubilize curcuminoids and affect cell uptake, such as bovine serum albumin (ITAYA et al., 2019). Whereas the susceptibility of bacteria to antibiotics is related to their metabolism, bacterial strains susceptible to antibiotics have a higher metabolic rate than antibiotic-resistant strains. The drug administered with a compound to metabolized may facilitate the absorption of the therapeutic compound. Glucose and fructose can increase the NADH production and protonmotor force due to the activation of the citric acid cycle (CAC). Thus, the use of a metabolite
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stimulates the incorporation of a drug, especially for resistant strains that have a slower metabolism (PENG et al., 2015).
The biological membrane is the main cellular component of interaction with amphipathic
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molecules such as curcumin, which has the main hydrophobic chain with some polar
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functional groups that are capable of interacting with the biological membrane and influences the fluidity similar to other amphipathic molecules such as cholesterol (SUN et al., 2008). The
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head of the phospholipids interacts with the methoxy or hydroxyl groups of curcumin — the direction of binding influences differently on the degree of order of the bilayer. The
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interaction of curcumin at low concentrations occurs only on the surface of the membrane with the ordered acyl groups; however, when the connection is perpendicular are disordered.
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Curcumin can present a steric hindrance in the binding region by promoting arrangements locally since the non-binding region can exhibit a disorder for entropic compensation
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(BARRY et al., 2009). This modulation of the membrane is similar to the mechanism of eukaryotic cells with the presence of cholesterol in the membrane; however, the bacterial mechanical stability is maintained by a cell wall, since cholesterol is absent. However, the effects of curcumin in the biologic membrane are not all understood yet. In addition to interaction with the phospholipids, curcumin can modulate the activity of membrane proteins as verified by INGOLFSSON et al., 2007 and HUNG et al., 2008 by studying the channels
formed by gramicidin A without affecting its conductance that modify the thickness and elastic property of the bilayers. Membrane integrity studies were performed with propidium iodide and calcein and verified that the protein has membrane damaging property (TYAGI et al., 2015). Bacterial cell death caused by PDT results from damage to the cytoplasmic membrane and DNA. Membrane proteins are modified by damage which alter concentration gradients and bacterial cell wall synthesis, factors that are sufficient to induce microbial death. The quantum
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efficiency may differ depending on location them hiding the diffusion of ROS in molecular targets (HAMBLIN; HASAN, 2004). However, internalization making the process more effective; it allows the action of the two pathways: type I (electron transfer) requires
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internalization; type II (energy transfer) is not necessary. Thus, the incubation times allow the localization of PS in the cell, which will aid in the diffusion of the reactive species and
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consequently the efficiency of the photodynamic action, especially for the type I reactions that
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should preferably occur in the cellular interior for greater efficacy, because there are larger biomolecules available for iteration (BACELLAR et al., 2015).
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There are several studies with different compounds of the pharmaceutical form which aim to aid the solubility of curcuminoids in water for clinical applications. Our results suggest that
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the interaction between curcuminoids may favor their solubilization and affect the interaction with the bacterial cell. In addition to PS uptake, it is important that the formulation is
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homogeneous to promote photodynamic inactivation. In the case of natural curcumin and DMC were the molecules with better solubility and homogeneity of the formulation compared to the synthetic and BDMC. Besides that, a bacterial community of multiple species has symbiosis relationships against external agents that explains the lower efficiency of aPDT. Appropriate curcumin uptake and understanding of the model presented here to assist the legitimate use of syrup in clinical application.
5. Conclusion The uptake of curcumin by bacteria is an essential step to an efficient aPDT. Besides, the physicochemical characteristics of the molecule and the uptake rate can be modulated by the pharmaceutical formulations, allowing an alternative strategy to implementation of treatments in clinical cases when the PS is more soluble in cytotoxic. Our results are suggesting that the use of curcuminoids in syrup favors the incorporation of this PS, mainly natural curcumin and DMC, improving the clinical protocols for antimicrobial control,
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allowing the use of lipophilic molecules as curcumin in aqueous solutions. Acknowledgement
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
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Superior - Brasil (CAPES) - Finance Code 001 and Center of Optics and Photonics (CEPOF-
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Fapesp 2013/07276-1). As well as CNPQ INCT program.
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Figures
Figure 1: Uptake of different concentrations of curcumin syrup, curcumin in alcohol/DMSO
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and aPDT for different concentrations of curcumin syrup incorporated into bacteria. A) uptake and B) aPDT of S. mutans. C) Uptake and D) aPDT of S. pyogenes. E) uptake and D) aPDT
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of clinical isolated from PT. *; **; ***, # indicates a statistical significance of p<0.05; 0.01;
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0.005 and 0.001, respectively.
Figure 2: Microbial grown curve (600 nm) with or without sucrose (syrup): A) S. mutans.
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B) S. pyogenes; C) clinical isolated of PT.
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Figure 3: Interaction of different curcuminoids (natural, synthetic, desmethoxycurcumin and bisdemethoxycurcumin) in syrup with S. pyogenes in 2.25 mg/ml. A) Rate of uptake.
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B) Reduction of log (CFU/ml) by aPDT with 60 J/cm².