Addition of hydrogen peroxide to methylene blue conjugated to β-cyclodextrin in photodynamic antimicrobial chemotherapy in S. mutans biofilm

https://doi.org/10.1016/j.pdpdt.2019.09.004Get rights and content

Highlights

  • Oral biofilm-dependent diseases can be treated with PACT.

  • PACT with β-cyclodextrin + methylene blue reduces S. mutans in single species biofilm.

  • Hydrogen peroxide behaved similarly as photosensitizers.

  • PACT with laser showed no statistical difference compared to chlorhexidine 0.2%.

  • The addition of hydrogen peroxide did not enhance the PACT effect.

Abstract

Objective

This study evaluated the effect of hydrogen peroxide addition on β-cyclodextrin-conjugated methylene blue in antimicrobial photodynamic therapy(a-PDT) in S. mutans biofilm model using laser or light emitting diode (LED) (λ = 660 nm).

Methods

A preliminary assay was performed to evaluate the cytotoxicity of hydrogen peroxide in oral fibroblasts by the colorimetric method (MTT). Afterwards, groups were divided into (n = 3, in triplicate): C (negative control), CX – chlorhexidine 0.2% (positive control), P (methylene blue/β-cyclodextrin), H (Hydrogen Peroxide at 40 μM), PH, L (Laser), LP, LH (Laser+Hydrogen Peroxide), LPH, LED, LEDP, LEDH, and LEDPH. The biofilm was formed in 24 h with BHI + 1% sucrose (w/v). Light irradiations were conducted with laser, 9 J, 323 J/cm2, 113 s or with LED, 8.1 J, 8.1 J/cm2 for 90 s. Microbial reduction was evaluated by counting the viable microorganisms of the biofilm after the respective treatments, in a selective culture medium, and laser confocal microscopy evaluation.

Results

LP, LH, LPH, LEDP, LEDH, and LEDPH groups statistically reduced the counts of S.mutans compared with the C group and the log reductions were of 1.87, 1.94, 2.19, 0.91, 0.92, and 1.33, respectively; the addition of hydrogen peroxide did not potentiate the microbial reductions (LPH and LEDPH) compared with the LP and LEDP groups.

Conclusion

The association of hydrogen peroxide with the conjugated β-cyclodextrin nanoparticle as photosensitizer did not result in an enhanced effect of a-PDT; hydrogen peroxide behaved as a photosensitizer, since it reduced the number of S. mutans when associated with laser light.

Introduction

Dental caries, a chronic disease prevalent in adults and children worldwide can be prevented if the microbiota associated with caries, such as Streptococcus mutans, Actinomyces, Lactobacillus, and Bifidobacterium species, is controlled on the tooth surface and, consequently, inhibit biofilm formation [1,2]. Another way is controlling sugar intake, since excessive and frequent sugar intake associated with poor oral hygiene is responsible for the substitution of the initial, nonpathogenic biofilm, for a shifted acidogenic and aciduric colonized biofilm [3]. However, controlling sugar intake in the population is rather difficult. Therefore, innovative therapies should be studied to discover new alternatives to control biofilm [4], since the used conventional methods are not always effective. Dental brushing depends on the patients’ collaboration [5], who often do not comply with a daily biofilm control consistent with good oral health.

In this perspective, photodynamic antimicrobial chemotherapy, which focus on the reduction of microorganisms, has been used as adjuvant therapy in the microbiological control of oral biofilms [6] and also as a complementary therapy after selective caries removal [[7], [8], [9]] with good perspectives in clinical use, including light sources such as lasers, LEDs and novel technologies in modern Dentistry [6].

A = PDT or PACT is based on the use of photosensitizers, molecules that absorb light and initiate a photochemical reaction when exposed to the light of specific wavelength. This process leads to the formation of reactive oxygen species, including singlet oxygen and free radicals that can cause irreversible damage to essential elements of bacterial cells, alter cell metabolism, and cause bacterial death [10,11] without affecting the host [12]. One advantage of PACT is that it can be an alternative to the use of chlorhexidine, since this antimicrobial agent shows limitations when in continued used [13] and also because it might develop drug resistance to the oral microorganisms [14].

Many studies have already shown the efficacy of PACT on oral bacteria, reducing bacterial load in cariogenic biofilms [[15], [16], [17], [18], [19]]. On the other hand, some researchers have shown that PACT has not been effective [[20], [21], [22], [23]], especially in the presence of sucrose, due to the bacterial capacity to use this carbohydrate to produce polysaccharides, turning this matrix-rich biofilm, difficult to be diffused by the photosensitizer [24]. To overcome this limitation and to potentiate the PACT effect, nanoparticles of β-cyclodextrin have been associated with photosensitive agents [[25], [26], [27]]. Such biocompatible association has been shown to increase bioavailability of photosensitizers, thereby increasing its potency and biological effect, due to the ability of the nanoparticle to solubilize small organic molecules [28].

A different way of potentiating PACT is the association of the photosensitizer with hydrogen peroxide (H2O2) to increase the death of microorganisms by two action mechanisms: 1) a chemical reaction between H2O2 and reactive oxygen species produced during the PACT that will improve the photosensitizer’s photochemistry or 2. due to the presence of H2O2 which will facilitate the photosensitizer diffusion inside the microbial cells [29]. Another hypothesis is that during irradiation, the medium oxygen pressure decreases [30] leading to oxygen depletion and consequent degradation of the photosensitization reaction. Therefore, if we increase the oxygen dissolved in the medium [31], for instance, in H2O2 form, the oxygen depletion could be avoided, and the PACT reaction could be potentiated. Hence, associating hydrogen peroxide with a photosensitizer (methylene blue conjugated to β-cyclodextrin) in antimicrobial photodynamic therapy may be effective in reducing the number of microorganisms in a S. mutans biofilm model.

Therefore, we aimed to evaluate the effect of the addition of hydrogen peroxide associated with methylene blue conjugated to β-cyclodextrin on photodynamic antimicrobial chemotherapy in S. mutans oral biofilm using red laser or red LED light sources (λ = 660 nm).

Section snippets

Experimental design

A preliminary assay was performed to evaluate the cytotoxicity of hydrogen peroxide in oral fibroblasts by the colorimetric method. S. mutans biofilms were produced in 96-well plates for 24 h. PACT was tested by the count of viable microorganisms in a selective medium and by laser confocal microscopy evaluation after the treatments, which were conducted in groups described in Table 1 (n = 3), in triplicate.

Cytotoxicity of hydrogen peroxide in human oral fibroblasts

The cytotoxicity of hydrogen peroxide (H2O2) was evaluated, in duplicate, in human oral

Results

The nanoparticle characterization in Fig. 1 shows the β-cyclodextrin encapsulated methylene blue in the concentration of 4.65 μM with defined particles and size around 230 nm.

Discussion

The capability of PACT to inactivate oral microorganisms in biofilm has been widely researched and has shown to be an effective therapy [36,37]. Considering the importance of cariogenic biofilm control, this study was designed to potentiate the effect of photodynamic antimicrobial chemotherapy, by means of adding hydrogen peroxide, on the reduction of S. mutans in biofilms by using two different PACT protocols, associating the photosensitizer and the nanoparticle with either laser or LED as

Support

This work was supported by the National Council for Scientific and Technological Development– CNPq (132211/2017-3); São Paulo Research Foundation – FAPESP (2017/03263-3); and the Coordination for the Improvement of Higher Education Personnel Foundation – Brazil (CAPES) – Finance Code 001.

Ethical approval

Informed consent form was obtained from participants included in the study under protocol # 65011017.9.0000.5418.

Declaration of Competing Interest

None.

Acknowledgements

The authors thank the valuable contributions of Dr. Flavia Sammartino Mariano Rodrigues and Marcelo Maistro for their technical support.

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