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hotodiagnosis and Photodynamic Therapy (2012) 9, 122—131

Available online at www.sciencedirect.com

jo ur nal homepage: www.elsev ier .com/ locate /pdpdt

EVIEW

ntibacterial photodynamic therapy for dentalaries: Evaluation of the photosensitizers used andight source properties

uliana Yuri Nagata MDa,∗, Noboru Hiokab, Elza Kimurac,agner Roberto Batistelab, Raquel Sano Suga Teradaa,riane Ximenes Gracianoa, Mauro Luciano Baessod,itsue Fujimaki Hayacibaraa

Dentistry Department, State University of Maringá, Maringá, Paraná, BrazilChemistry Department, State University of Maringá, Maringá, Paraná, BrazilPharmacy and Pharmacology Department, State University of Maringá, Maringá, Paraná, BrazilPhysics Department, State University of Maringá, Maringá, Paraná, Brazilvailable online 23 December 2011

KEYWORDSPhotodynamictherapy;Dental caries;Photosensitizer

Summary Photodynamic therapy studies have shown promising results for inactivation ofmicroorganisms related to dental caries. A large number of studies have used a variety of pro-tocols, but few studies have analyzed photosensitizers and light source properties to obtainthe best PDT dose response for dental caries. This study aims to discuss the photosensitizersand light source properties employed in PDT studies of dental caries. Three questions wereformulated to discuss these aspects. The first involves the photosensitizer properties and theirperformance against Gram positive and Gram negative bacteria. The second discusses the useof light sources in accordance with the dye maximum absorbance to obtain optimal results.The third looks at the relevance of photosensitizer concentration, the possible formation ofself-aggregates, and light source effectiveness. This review demonstrated that some groups ofphotosensitizers may be more effective against either Gram positive or negative bacteria, thatthe light source must be appropriate for dye maximum absorbance, and that some photosensi-tizers may have their absorbance modified with their concentration. For the best results of PDTagainst the main cariogenic bacteria (Streptococcus mutans), a variety of aspects should betaken into account, and among the analyzed photosensitizer, erythrosin seems to be the most

appropriate since it acts against this Gram positive bacteria, has a hydrophilic tendency andeven at low concentrations mayappropriate light source should530 nm, which may be achieved© 2011 Elsevier B.V. All rights re

∗ Corresponding author at: Rua Irmã Eleotéria, 670, CEP: 86800-300, Ael.: +55 43 34225996; fax: +55 43 34225996.

E-mail addresses: ju nagat@hotmail.com (J.Y. Nagata), nhioka@uem.agner.batistela@yahoo.com.br (V.R. Batistela), rssterada@uem.br (R.S.lbaesso@uem.br (M.L. Baesso), mfhayacibara@uem.br (M.F. Hayacibar

572-1000/$ — see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.pdpdt.2011.11.006

have photodynamic effects. Considering erythrosin, the most

have a maximum emission intensity at a wavelength close to

with low cost LEDs.served.

pucarana, Paraná State, Brazil.

br (N. Hioka), ekimura@uem.br (E. Kimura),S. Terada), arianeximenes@hotmail.com (A.X. Graciano),a).

Photodynamic therapy for dental caries 123

Contents

Introduction.............................................................................................................. 123Photodynamic therapy for dental caries.................................................................................. 123Which photosensitizers are more effective against specific groups of dental caries bacteria?............................. 123Are the light sources appropriate for the different dyes? ................................................................. 126How does the dye concentration influence the effectiveness of the therapy?............................................. 127Future developments..................................................................................................... 129Conclusions .............................................................................................................. 129References ............................................................................................................... 129

Introduction

Dental caries is one of the most prevalent chronic diseasesin the population worldwide, affecting 60—90% of school-aged children and almost 100% of the adult population [1].The prevalence of dental caries has been studied in manydeveloped countries in recent years. In the USA, caries wasconsidered the most common chronic disease of childhood,being five times more common than asthma [1], with aprevalence of 27% in preschoolers, 42% in school-aged chil-dren, and 91% of dentate adults [2].

Dental caries results from interactions over timebetween specific pathogenic bacteria, primarily Streptococ-cus mutans, which metabolize ingested carbohydrates toform acids [3,4]. In recent decades, photodynamic therapy(PDT) has been studied as an alternative measure againstthe etiological factors of dental caries. PDT is a treatmentthat utilizes light to activate a photosensitizing agent in thepresence of oxygen, resulting in the production of reactiveradicals capable of inducing cell death [5].

In the literature, there is a large number of studiesshowing a variety of protocols for the use of PDT, butonly a few of them analyze the properties of the pho-tosensitizers and light sources used in Dentistry in orderto obtain the best dose response of photodynamic ther-apy for dental caries. Currently, PDT is being appliedmostly in the treatment of macular degeneration, patho-logical myopia, esophagus, lung, and skin cancer, and inthe treatment of precancerous lesions in Barret esophaguspatients [6]. Additionally, several studies have shown thatPDT also has antimicrobial properties, in a process called‘‘photodynamic inactivation’’ (PDI) or ‘‘photodynamicantimicrobial therapy’’ (PACT) or even ‘‘Photo-activateddisinfection’’ [7—12]. These antimicrobial properties havebeen extended and studied for the treatment of caries[13—21].

Several groups of photosensitizers in different illumi-nation systems have been proposed. Even when the samephotosensitizer (PS) and light source were employed, thediversity of irradiation protocols and variation of PS con-centration, irradiation time, and light potencies makescomparison between the results difficult. Few studies dis-

Photodynamic therapy for dental caries

The key-words photodynamic therapy; S. mutans, and den-tal caries were entered in Medline, Bireme, and Scielodatabases, resulting in 18 articles related to PDT and dentalcaries (Table 1). These studies involved in vitro and in situexperiments with a variety of PDT protocols that were pub-lished between 1992 and 2010. Based on these variations,three questions were formulated in order to discuss the useof different types of photosensitizers in different concen-trations and the influence of the light sources on the resultsof these PDT researches.

Which photosensitizers are more effectiveagainst specific groups of dental cariesbacteria?

The main organisms recognized as associated with earlycaries development are the Streptococci mutans group (par-ticularly, S. mutans and S. sobrinus) and lactobacilli species[29]. As the lesion progresses to deeper dentin, anaerobicspecies start to thrive and a transition takes place frompredominantly facultative Gram positive bacteria to strictlyanaerobic Gram positive rods and cocci, and Gram nega-tive rods [30]. In the analyzed articles, S. mutans was themost studied bacteria, since they are the main specimenrelated to dental caries. Moreover, other specimens werealso evaluated by these authors, such as Lactobacillus aci-dophilus, Streptococcus sobrinus, Streptococcus sanguinis,and Actinomyces naeslundii.

From the point of view of bacteria and PS interaction,the effectiveness of PDT is mostly related to three mainaspects: (a) photosensitizer capability of interacting withthe bacterial membrane; (b) photosensitizer ability of pen-etration and action inside the cell, and (c) reactive singletoxygen formation around the bacterial cell by illuminationof the PS.

The cell membrane binding mechanism is different inGram positive and Gram negative bacteria. This differencecan be explained by structural variations in their cell walls,and hydrophobic and charge effects of the photosensitizers.

cuss both the structural properties of PS and of thelight sources to specifically achieve the optimal proto-col of this therapy against dental caries. Therefore, thisstudy aims to discuss the properties of photosensitizersand light sources employed in PDT studies for dentalcaries.

Gwamit

ram negative bacteria present a complex outer membranehich includes two lipid bilayers that work as a physical

nd functional barrier between the cells and the environ-ent, while Gram positive cells have a thick membrane that

s relatively permeable [31]. It is possible that this rela-ively porous layer of peptidoglycan and lipoteichoic acid

124

J.Y. N

agata et

al.

Table 1 Studies about antibacterial photodynamic therapy for dental caries.

Study Microorganism Photosensitizer Light source Dose energy

Burns et al. [22] In vitro (Streptococcus mutans) TBO and aluminumdisulphonated phthalocyanine(AlPcS2)

HeNe LASER (633 nm) or GaAlAsLASER (660 nm)

0.876—3.504 J/cm2

0.01—0.752 J/cm2

Wilson et al. [11] In vitro (Streptococcus sanguis) AlPcS2 (0.1 mg/mL) GaAlAs LASER (660 nm) 4.1 J/cm2

Wood et al. [23] Biofilm in situ Pyridinium Zn(II)phthalocyanine (PPC)(0.02 mg/mL)

Tungsten filament lamp whitelight (600—700 nm)

40.5 J/cm2

Williams et al. [14] In vitro (Streptococcus mutans) TBO (13 mg/mL) Diode LASER (633 nm) 2—288 J/cm2

Paulino et al. [15] In vitro (Streptococcus mutans and fibroblasts) Rose bengal (0—0.05 mg/mL) Halogenlamp-photopolymerizer(400—500 nm)

0.35—0.5 J/cm2

Zanin et al. [24] In vitro (Streptococcus mutans) TBO (0.1 mg/mL) Diode laser (632.8 nm)/HeNelaser (638.8 nm)

49—294 J/cm2

Metcalf et al. [25] In vitro (Streptococcus. mutans) Erythrosin (0.019 mg/mL) White light (500—550 nm) 6.75 J/cm2

Wood et al. [10] In vitro (Streptococcus mutans) Erythrosin (0.019 mg/mL),PhotofrinTM (0.026 mg/mL) andMB (0.007 mg/mL)

Tungsten filament(500—550/600—650 nm)

20.43 J/cm2

20.25 J/cm2

Hope and Wilson [26] In vitro (Streptococcus pyogenes) Sn IV Chlorin e6 (0.05 mg/mL) HeNe (543 nm)/Argon laser(488 nm)

6.75 J/cm2

Zanin et al. [16] In vitro (Streptococcus mutans/Streptococcussobrinus/Streptococcus sanguinis)

TBO (0.1 mg/mL) Red LED (638.8 nm) 85.7 J/cm2

Müller et al. [27] In vitro (Actinomyces naeslundii/Veillonelladispar /Fusobacteriumnucleatum/Streptococcussobrinus/Streptococcus oralis/Candidaalbicans)

MB Diode LASER (665 nm) NR

Bevilacqua et al. [17] In vitro (Streptococcus mutans) TBO (0.1 mg/mL) LED (640 nm) 2.18 J/cm2

Giusti et al. [18] In vitro (Streptococcus mutans andLactobacillus acidophilus)

PhotogemTM (1, 2, 3 mg/mL)and TBO (0.025, 0.1 mg/mL)

Red LED (630 nm) 24 J/cm2

48 J/cm2

Maisch et al. [19] Streptococcus mutans; Enterococcus faecalisand Aggregatibacter actinomycetemcomitans

PhotosanTM (0.0005; 0.001;0.005; 0.01; 0.05; 0.1 mg/mL)

Blue LED (450 nm) 9.65 J/cm2

Lima et al. [20] In situ (Streptococcus mutans) TBO (0.1 mg/mL) Red LED (638.8 nm) NRAraújo et al. [28] In vitro (Streptococcus mutans) TBO and MB (0.025; 0.01 and

0.005 mg/mL)Red LASER NR

Bolean et al. [21] In vitro (Streptococcus mutans) Rose Bengal (0.00009 mg/mL) Halogenlamp-photopolymerizer(400—500 nm)

0.35—0.5 J/cm2

Vahabi et al. [11] In vitro (Streptococcus mutans) TBO (1 mg/mL) andRadachlorinTM (1 mg/mL)

Laser (662 nm) 12 J/cm2

NR, not reported.

oTIomnaqtdctabTenacaZawptdoaPattcMumtttt

riscaea(mtt[[bite

Photodynamic therapy for dental caries

outside the cytoplasmic membrane of Gram positive speciesallows the photosensitizer to diffuse into sensitive sites [5].In general Gram negative species are significantly resistantto some commonly used photosensitizers in PDT [31]. In thisreview of the literature (Table 1), all the papers evaluatedand obtained effective PDT results against Gram positivespecies, which are more susceptible, because they have noprotective external membrane.

Besides these structural differences, photosensitizercharge may influence the inactivation of Gram positiveand Gram negative specimens. In general, it is assumedthat at physiological pH, neutral or anionic compounds,such as rose bengal, erythrosin, eosin, porphyrin derivatives(PhotofrinTM, PhotosanTM, and PhotogemTM), and aluminumdisulphonated phthalocyanine bind efficiently and inac-tivate Gram positive bacteria, while in Gram negativebacteria, these photosensitizers bind to the outer mem-brane to some extent but do not effectively inactivate themafter illumination. Eight of the 18 selected articles (Table 1)studied anionic photosensitizers, depending on their pH insolution (rose bengal, erythrosin, PhotofrinTM, PhotogemTM,PhotosanTM, and aluminum disulphonated phthalocyanine)against Gram positive bacteria. In these studies, low concen-trations of rose bengal were capable of totally eliminatingbacteria [14] and caused stressing conditions unfavorableto cell viability [21]. For erythrosin, good results were alsoobserved with different light sources and irradiation time[25]. When compared with methylene blue (MB), inactiva-tion by erythrosin was better [10]. However, in other studies,when these photosensitizers were compared with erythrosinand toluidine blue O (TBO), the latter resulted in signifi-cantly higher bacterial reduction [10,18].

In contrast, cationic photosensitizers, such as MB, TBO,and pyridinium Zn(II) phthalocyanine, are capable of inac-tivating both Gram positive and Gram negative bacteria.The resistance of Gram negative bacteria against efficientkilling by anti-bacterial photodynamic therapy is due tothe different outer membrane structures of Gram posi-tive and Gram negative bacteria [32]. These positivelycharged dyes may bind to the polyphosphates of the outermembrane and produce molecular damage to lipids and pro-teins, including membrane-bound enzymes [33]. Anothermechanism proposed for these positively charged PS is thecrossing of the membrane and consequently the attractionto the negatively charged potential of mitochondria, whichallows direct action on this organelle [34—36]. The articleswhich used cationic photosensitizers, such as TBO, MB, andpyridinium Zn(II) phthalocyanine, against Gram positive bac-teria obtained significant inhibition of the target bacteriawith different concentrations and irradiation sources andtimes. One study compared two anionic dyes (erythrosinand PhotofrinTM) with one cationic dye (methylene blue)and observed that erythrosin was 1—2 log more effective atkilling biofilm bacteria than PhotofrinTM and 0.5—1 log moreeffective than MB [10].

Another PDT mechanism is the photosensitizer penetra-tion and action inside the cell. This is possible due to thehydrophilicity and solubility of the dyes, which determine

how readily they cross the cellular wall [33]. Gram posi-tive bacteria protect their cytoplasmic membrane with athick multilayer peptidoglycan wall that blocks the pas-sage of hydrophobic components because of the presence

trgo

125

f amino acids and sugars within the cell membrane [37].herefore, only hydrophilic components penetrate this wall.

n contrast, Gram negative bacteria have one or a few layersf peptidoglycan and an external membrane. Because thisembrane presents lipoproteic character, special mecha-

isms such as the passage through pores are necessary tollow the intake of hydrophilic components [37]. Conse-uently, hydrophobic components are expected to penetratehe cell better than hydrophilic ones. In general, theyes may either have a more hydrophilic or hydrophobicharacter or may be amphiphilic. The literature considershat rose bengal, MB and TBO present amphiphilic char-cter (both hydrophobic and hydrophilic), of which roseengal has a more hydrophobic character, and MB andBO have a more hydrophilic character [36]. In addition,rythrosin is considered hydrophilic, and phthalocya-ine and porphyrin derivatives (PhotofrinTM, PhotogemTM,nd PhotosanTM), hydrophobic. In the selected arti-les, hydrophobic (PhotofrinTM, PhotogemTM, PhotosanTM,luminum disulphonated phthalocyanine, and pyridiniumn(II) phthalocyanine (PPC)), hydrophilic (erythrosin), andmphiphilic (MB, TBO and rose bengal) photosensitizersere studied. Since all the investigated bacteria were Gramositive, better results were expected with hydrophilic pho-osensitizers. Nevertheless, papers that tested hydrophobicyes also reported good results with significant inhibitionf the target bacteria [10,13,18,19,22]. Two articles usedmphiphilic (TBO and MB), hydrophobic (PhotofrinTM andhotogemTM), and hydrophilic (erythrosin) photosensitizers,nd in both studies, the hydrophilic dye was more effectivehan the amphiphilic and hydrophobic ones in the inactiva-ion of Gram positive S. mutans [10,18]. In addition, theomparison of two amphiphilic photosensitizers (TBO andB) resulted in higher bactericidal efficacy for TBO. Its sol-bility should be higher in the hydrophobic region of theembrane, and thus TBO should interact more easily with

he bacterial membrane than MB [33]. Considering the facthat erythrosin is the most hydrophilic photosensitizer andhat the Gram positive bacteria allow better penetration ofhis dye, it seems to be the most appropriate for PDT.

Besides the photosensitizer capacity to bind to the bacte-ial membrane and penetrate bacteria, there are reports ofnactivation of bacteria, in which it is clear that the photo-ensitizer does not have to penetrate or even to come intoontact with the cells to be effective. According to someuthors, if sufficient quantities of singlet oxygen can be gen-rated near the outer membrane of the bacteria, it will beble to inflict damage on vital structures [38]. The lifetime�) of singlet oxygen is highly dependent on the environ-ent, and when in solution, it varies with the nature of

he solvent. In water, its lifetime (�) is around 4 �s, and inhe biological systems, it is extremely low, less than 0.04 �s39], which reduces its radius of action to about 0.02 �m40]. Therefore, if the PS cannot interact with the targetacteria, but the singlet oxygen is generated in close prox-mity to the cell, its viability will depend on the distance tohe bacteria. Therefore, reaching the expected therapeuticffect does not necessarily involve total affinity between

he bacterial wall and the PS. It is also important that theeactive products of therapy (such as singlet oxygen) areenerated, because success may be achieved even with-ut direct contact between the PS and the bacteria. The

126 J.Y. Nagata et al.

Table 2 Three main groups of PDT light sources and their characteristics.a

Type of light source Laser LED Halogen lamps

Efficiency (single optical fibers) High (>90%) Low (25—50%) High (>50%)Cost High Low HighWavelength Single Region (±30 nm) Broad (400—500 nm)Heating Yes Yes Yes

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uantity of singlet oxygen generated by the photodynamiceaction varies for each dye. This quantity is measured by itsinglet oxygen quantum yield. Literature reports the oxygenroduction and the quantum yield values from the lowesto the highest for phthalocyanine derivatives (0.56), methy-ene blue (0.59), toluidine blue o (0.60), erythrosin (0.63),ose bengal (0.76), and haematoporphyrins derivatives, suchs PhotofrinTM (0.83) [41]. The higher the quantum yield, theigher the production of singlet oxygen, and consequently,he higher the photodynamic efficacy. However, in biologicalystems several factors can affect this activity.

Therefore, the choice of an appropriate photosensitizerust consider all these aspects. The first factor is the species

f the target bacterium. If it is Gram positive, both cationicnd anionic dyes may be utilized, and if it is Gram negative,ationic dyes are more effective. Concerning the penetra-ion of the PS in the bacteria, the water solubility of theye must also be taken into account, since hydrophilic pho-osensitizers have a higher penetration in Gram positiveacteria, while hydrophobic dyes have a higher penetrationn Gram negative bacteria. Moreover, if toxic and reactiveroducts of the PDT can be created near the bacterial mem-rane, effective inactivation may be observed, even withouthe direct bacterium-photosensitizer contact. Thus, amonghese aspects, the most relevant in the choice of a pho-osensitizer seems to be the structural characteristics ofhe bacterial membrane (Gram positive or negative), sincet would lead to some dyes having a more effective toxicction.

re the light sources appropriate for theifferent dyes?

he basic requirement for PDT light sources is that theyatch the activation spectrum (electronic absorption spec-

rum) of the photosensitizer (usually the longest wavelengtheak) and generate adequate light potency at this wave-ength [42]. This concept was first discussed by Isaac Newtonn 1666, when he showed that the light colors red, orange,ellow, green, blue, and violet together compose whiteight. Later, Newton presented ‘the Newton disc’, showinghat the rotation of a disc painted with the colors that com-ose white light resulted in the observation of a white disc.n the mid 19th century (1853), the Polish mathematicianrassman published a theory on the concept of complemen-ary light, showing that each color of the Newton disc has

nother complementary color in the same disc [43]. Fur-hermore, at the end of the 19th century, Ewald Heringeveloped the opponency theory, in which he revealed thatreen is the opposite color of red, and blue of yellow. This

6ta(

s in accordance with the PDT, in which the photosensitizerhould have the maximum absorbance achieved by an appro-riate and complementary light source; for example, a bluehotosensitizer must be irradiated by a red light, which isore absorbed, both of which are complementary colors

43].The literature presents three main classes of clinical

DT light sources: LASER, LED and halogen lamps (Table 2).ASER has some advantages, such as monochromaticity andigh efficiency (>90%) of coupling into single optical fibersn endoscopic, high potency, and interstitial light deliveryevices; however, they do have a high cost. Diode LASERs one of the lowest priced among LASER systems. It is veryonvenient and reliable; however, it has a single wavelengthnd requires a separate unit for each photosensitizer dueo the different absorption wavelengths. LED has become

viable technology for PDT in the last few years, particu-arly for irradiation of easily accessible tissue surfaces. Theain advantages of LED over LASER or diode LASER sources

re their low cost and ease configuration of LED arrays intoifferent irradiation geometries. As with LASER diodes, LEDave a fixed output wavelength, but as the cost per watts significantly smaller, having different sources for eachhotosensitizer is less of a drawback [42]. Filtered halogenamps have the advantage that they can be spectrally fil-ered to match any photosensitizer; however, they cannot befficiently coupled into optical fiber bundles or liquid lightuides, and also cause heating. With broadband sources,heir effective output potency is lesser, as compared to aASER source at the photosensitizer activation peak, and it isroportional to the integrated product of the source outputpectrum and the photosensitizer activation spectrum. ForED and filtered halogen lamps, the output spectrum typi-ally has a bandwidth of about 25—30 nm and an efficiencyactor for the typical photosensitizer spectrum of about 50%42].

The selected articles (Table 1) show results obtainedsing different light sources such as LASER, LED, halogenamps, and tungsten filaments. Most of the papers employedhenotiazinic dyes, such as TBO and MB, associated withed light (LASER or LED) [10,11,14,16—18,20,22,24,27,28].he maximum absorbance of these components occurs at00—660 nm, and all the found articles used appropriateight sources (red color light). Other blue/green photosen-itizers used were the porphyrin derivatives (PhotofrinTM,hotogemTM, and PhotosanTM) and phthalocyanine deriva-ives, which have the maximum absorbance for red light at

30 nm and at 600—700 nm, respectively, which correspondso the porphyrin Q band. In these studies, PhotogemTM

nd PhotofrinTM were successfully irradiated with red LED630 nm) and tungsten filament (600—650 nm), respectively

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Photodynamic therapy for dental caries

[10,18]. For the phthalocyanine derivatives, light sources(tungsten filament, HeNe, and GaAlAs LASER) compati-ble with the maximum absorbance of the dyes were alsoemployed with success [11,22,23]. In contrast, for thePhotosanTM, a blue LED light source with wavelength of450 nm was used in the porphyrin Soret band [19]. The goodresults obtained with this association could, nevertheless,be improved if another light source with a more appropri-ate wavelength were used, because irradiation at 450 nmpresents lower biological tissue light penetration as com-pared to the 600—700 nm region.

Other photosensitizers reported in the articles were red-colored, such as rose bengal and erythrosin, which absorb at561 and 530 nm, respectively. For rose bengal, the employedlight sources emitted wavelengths between 400 and 500 nm[15,21]. In these cases, improved effects would occur nearthe wavelength of 561 nm. Better results could be expectedif another light source were employed. On the other hand,for erythrosin, the articles utilized white light sources withemitting wavelength of 500—550 nm, which is in accordancewith the maximum absorbance of this dye [25].

The PDT is classically employed for some medical treat-ments which require efficient light penetration to reach thetarget lesion. Therefore, the absorption spectrum of PS mustbe between 600 and 800 nm for an efficient penetration ofradiation. The absorption coefficient of the majority of theirrigated tissues is determined by the concentration of light-absorbing molecules (chromophores). For PDT wavelengths,the two most important chromophores are hemoglobin andwater [42]. Considering this fact, the appropriate light colorin these cases is red, which corresponds to light penetrationfrom 0.5 cm (at 630 nm) to 1.5 cm (at 700 nm) [43]. Nev-ertheless, in dental caries, the hemoglobin chromophoremay be absent, and consequently, blue and green lightwould present no interference effect. Although most of theselected articles employed red light sources, the use of bluelights began to be studied, and further studies are neces-sary for a better understanding of tissue interference in theefficiency of PDT.

Few of the selected articles investigated the effect oferythrosin and fuchsin (red-colored compounds) as photo-sensitizers, and even fewer used halogen light or blue LEDas light sources. Both photosensitizers and light sources arepresent in the dental routine and can be used in PDT with-out requiring acquisition of new equipment. Similarly, noevidence was found of the association of these dyes withthe light sources used to activate resin composites. Furtherstudies employing dental routine materials, including dyessuch as erythrosin, malachite green, and fuchsin, and lightsources such as halogen lamps and LED, are necessary andmay facilitate the inclusion of PDT in the dental practice.

Related to the dose of energy studied in the articles agreat variation in illumination protocols could be noted,with different potencies and exposure times. The highestpotency observed in the articles (Table 1) refers to LASERsources [22,24], probably because this light concentratesgreat energy in a small area, and the longer irradiation timewas adopted for tungsten filament lamps [10,24], since this

light source has broad spectrum and low intensity requiringlonger time interval to achieve an efficient energy emis-sion. With compensations (potency and period of irradiationtime), LASER and tungsten lamps presented high dose of

toba

127

nergy. These aspects should be carefully analyzed whensed in teeth, since temperature increase in dentin and pulpissue may cause irreversible changes. Literature shows thatn increase in temperature of 2.2 ◦C resulted in no adverseffects, and an increase of 5.5 ◦C resulted in pulpal necro-is in 15% of cases. In addition, it was shown that when theemperature in the pulp increased about 11 ◦C, it routinelyuccumbed [44]. Irradiation with some light sources mayroduce temperature increase proportionally to the expo-ure time. The temperature increases in dentin producedy LED and halogen lamps have been studied. The use ofED showed no marked increase in temperature after 45 and0 s of exposure, while halogen lamps caused changes, with

maximum rise of 5 ◦C [45,46]. Some of the studied articleshowed longer period of exposure time (10, 15 and 30 min)10,23,24] using a tungsten filament lamp and LASERs. Theyhowed that this may cause a risk to dentin and pulp tis-ues, since long periods of exposure time proportionallyncrease the temperature of the irradiated area [46]. There-ore, thinking about the use of photodynamic proceduresor the inactivation of S. mutans, the use of LED may beuggested, considering its capacity of not changing the tem-erature allied to its high dose energy supply. In associationith LED, the most appropriate photosensitizer would varyccording to the spectrum of wavelength of the selectedED. With respect to continuous light (cw) or pulsed lightources, there are still controversial points of view and noonclusions. For high PDT efficiency, the presence of normalxygen close to the PS and the target tissue is necessary.y illumination, part of the oxygen is transformed in singletxygen, however for the continuation of the reaction moreegular oxygen is necessary. Usually, tissue such as that of

tumor exhibits a high blood supply that keeps oxygen at aigh level in the cell environment (such as membranes). Ift is not the case, pulsed light is interesting for at least, twoeasons: (i) it allows recovery of the level of regular oxygen;ii) allows the temperature of dental structures to be keptt acceptable levels.

ow does the dye concentration influence theffectiveness of the therapy?

DT may be classified inside the photophysical and pho-ochemical studies. A common problem found in this kindf study is the formation of dye self-aggregates in aque-us media, as aggregation usually impairs the therapeuticesponse of PDT [47]. The formation of aggregates modi-es the absorption spectrum and photophysical propertiesf the dye and affects its ability to absorb at a certain wave-ength or to act as a photosensitizer [48]. In addition, in theggregate state the PS may undergo a process called energyelf-quenching, diminishing the PS excited state form, whicheduces the singlet oxygen production [48]. The aggregationenerally occurs when the concentration of the dye, espe-ially of hydrophobic dyes, increases, with reactions in thequeous media between their monomeric forms to generateimers or higher self-aggregated forms. In low concentra-

ions, the monomeric form predominates and the spectrumf the dye generally presents its predominant absorptionand in the UV—vis spectrophotometry. On the other hand,s the dye concentration increases, an additional band may

1

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28

ometimes appear and the absorption of the dye may beodified. In general, dyes aggregate more strongly in water

han in organic solvents, and more generally still in solutionsf high ionic strength (presence of high level of salts).

This review focuses on the influence of dye concen-ration, the possible formation of aggregates, and thenterference in the dye absorption spectra. As observedarlier, the most studied dyes are MB and TBO in severaloncentrations. Both TBO and MB, in high concentrationsresent changes in the absorption spectrum due to electro-tatic and hydrophobic alterations in the dye molecules anddjacent molecules. This interaction results in dye aggrega-ion, with formation of dimers and oligomeric forms. Thisrocess generally decreases the peak of maximum absorp-ion of the monomer and increases the peak of the dimers,hich, in the case of MB and TBO, present lower wave-

engths [49,50]. The role of monomers and dimers in theethal photosensitization is not clear, but their wavelengthsf maximum absorption are different. For MB, the wave-ength of maximum absorption is 660 nm, whereas for itsimer form it is 610 nm. For TBO, the wavelength of maxi-um absorption in the monomeric form is 630 nm, whereas

or the dimeric form this wavelength decreases to 590 nm50]. By changing the wavelength of the irradiation source,t is observed that both species can be effective in micro-ial inactivation [50]. The photochemistry of MB monomersnvolves increased production of singlet oxygen (Type IIechanism, energy transfer reaction), while the dimers

ave a higher yield for Type I reactions (involving electronransfer reaction). Therefore, the two types of reactions cane important for the photodynamic process [44]. The lit-rature also shows that in concentrations up to 30 �mol/L10 �g/mL to 0.01 mg/mL) of TBO, the dimer band con-ributes more than the monomer band [34,50]. Among thenalyzed articles [14,16—18,20,22,24,28], only the concen-ration of 0.005 mg/mL did the monomer band predominatend are appropriated for the investigated wavelengths630 nm). Higher concentrations of this dye would be bet-er activated by light sources of 630 and 590 nm, sinceoth monomer and dimer bands would be present, withredominance of the dimeric form. For MB, the maximumbsorbance of the monomeric form happened at 660 nmntil the concentration of 10−6 mol/L (0.0003 mg/mL). Whenigher concentrations were employed, the dimers began toanifest at a wavelength of 610 nm. The selected articles

10,28] employed concentrations in which both monomericnd dimeric forms were present, with predominance ofimers. As can be seen, only one study used an appro-riate light source for the dimer solution (600 nm), whichorresponded to maximum absorbance. However, the idealituation would be the use of light sources emitting twoavelengths (660 and 610 nm) that acted on both monomersnd dimers.

In the case of fluorescein, eosin, erythrosin and roseengal, there are reports of formation of trimers andigh aggregates in aqueous solution [48]. For rose ben-al, the monomeric form existed up to a concentrationf 5 × 10−5 mol/L (0.05 mg/mL), which presented maximum

bsorbance at the wavelength of 550 nm [48]. When theoncentrations were higher, the absorption spectrum of theimers became visible at 490 nm. The studies [15,21] uti-ized low concentrations with predominance of monomers,

mhfi

J.Y. Nagata et al.

hich requires light sources of 550 nm. Nevertheless, themployed irradiation wavelength varied from 400 to 500 nm,hich seemed inappropriate for this solution, despite theood results found. For erythrosin, at concentrations lowerhan 5 × 10−6 mol/L, the absorption spectra are identi-al for monomers with maximum absorption at 530 nm.etween 5 × 10−3 and 10−4 mol/L, the spectra start toxhibit a general broadening and a slight blue shift withespect to the monomer spectra. At a higher concentra-ion (2.5 × 10−3 mol/L) there was evidence of oligomerictructures formation manifested by the blue shift. This waseinforced by the formation of a second band at 510 nm [51].t the concentration employed in the studies (0.019 mg/mL)here are both monomeric and oligomeric forms, with pre-ominance of monomers. Consequently light sources withavelengths in accordance with the maximum absorbancef the dye in the spectral range interval (500—550 nm) wastilized. For eosin, erythrosin and rose bengal (all halo-anthenes dyes), the best pH during illumination should keept physiological region, pH around 7.4, where the dianionicrotolytic form of this species prevails [52].

In the case of phthalocyanine derivatives, there is atrong tendency to form inactive dimers and higher aggre-ates. This process is favored in aqueous suspension dueo the highly hydrophobic core of the dye. The literatureeports that the derivative AlPcS2 does not aggregate atoncentrations between 0.25 and 7.0 × 10−6 mol/L and theredominance of the monomer occurs presenting maximumf absorption around 660 nm [51]. Higher concentrationsenerally show a second band relative to the dimers in theavelength of 600 nm [53]. The papers that utilized this dye

ested higher concentrations with predominance of dimers.lthough in these concentrations both forms of the dyere present, only one study used an appropriate irradiationource (600—700 nm) with a broad spectrum of emission.

The porphyrin derivatives (PhotofrinTM; PhotogemTM andhotosanTM) in general present a monomer absorbance bandn the wavelength of 630 nm up to the concentration of.8 × 10−6 mol/L [54]. When the concentration of the dye inolution exceeds this value, a second band in the region of00 nm appears, changing the maximum absorbance of thehotosensitizer. In the articles analyzed [10,17,18] it can beoted that the concentrations of 0.0005 and 0.001 mg/mLresented only monomeric forms; however, these studiesid not use the most suitable light source (450 nm) [19].or the highest concentrations, both monomers and dimersre present in the solution, which requires the irradiationith light sources of 630 and 600 nm wavelengths. Thisappened in the study of Wood et al. [10], in which theavelength utilized varied from 600 to 650 nm with solu-

ions of photosensitizers with both monomeric and dimericands (0.026 mg/mL). The other studies used light sourcesith maximum absorbance for the monomers, despite theresence of dimers in the solution.

A summary of the maximum absorption and concentrationf the analyzed dyes is shown in Table 3. Considering thespects discussed, the selection of a photosensitizer mustake into account its photophysical characteristics to deter-

ine the appropriate concentrations of study. In addition,

igher concentrations may require light sources with dif-erent wavelengths, according to the absorption alterationsnduced by the aggregation properties of the dye.

Photodynamic therapy for dental caries 129

Table 3 Maximum absorption and concentration of photosensitizer solution on the monomeric and aggregated forms.

Photosensitizer Absorption ofmonomers (nm)

Maximum concentration with predominanceof monomers (mg/mL)

Absorption ofaggregates (nm)

TBOa 630 0.01 590MBa 660 0.0003 610Erythrosinb 530 0.004 510Rose Bengalc 550 0.05 490Phthalocyanined 660 0.0000007 600Porphyrine 630 0.002 600

a Bergman and O’konski [49]; Usacheva et al. [50].b Stomphorst et al. [51].c Valdes-aguilera and Neckers [48].d Dhami and Phillips [53].e Pasternack et al. [54].

C

Tlisptsssasatnlm

R

Despite all above considerations, the main PDT treat-ment is considered a Type II mechanism, via singlet oxygenas a reactive specie that induces biological cellular dam-age [53,55]. Therefore, the use of self-aggregated dyes asphotosensitizers usually should be avoided. It is hoped thefuture clinical use of this therapy in Dentistry, since the bestapplication include low dye concentrations which providelow toxicity, high solubility, and unlikely dental staining.

Future developments

PDT will not replace classic therapy for dental caries, how-ever, the photodynamic approach may improve, accelerateand lower the cost of treatment, in addition to acting as anextra-protective measure for dental care in the medium tolong terms. Some of the advantages that may direct futureinsertion of this therapy include its antibacterial property,which may reduce dental structure removal during dentalcaries treatment, the low concentration of the dyes thatresult in low toxicity, high solubility and unlikelihood ofdental staining. Moreover, studies about the photodynamicproperties of dyes used in routine Dentistry such as dis-closing plaque materials including erythrosin, fuchsin andmalachite green may favor their clinical application. Fur-thermore, in routine dental practice, light sources are used(such as halogen lamps and LEDs for light polymerization ofcomposite resins) which, at the same time, are the necessarybasis/components for the application of PDT. In addition,some of the dyes found in dental offices, such as erythrosinand others that have shown proof of generating a high levelof singlet oxygen, exhibit excellent photodynamic proper-ties. With regard to the association (PS-light source), themaximum absorbance of red-colored dyes may be comple-mentary to some blue-colored light sources, however eachindividual property should be carefully studied before itsapplication, because of the need to match the emission

spectra of light and absorption spectra of light with the PS.Thus, some articles, would perhaps have found better resultsif some of these aspects had been taken into account, sincethese factors must be always considered in PDT studies.

onclusions

he real mouth environment is totally different from theaboratorial culture or in vitro environment, which makest difficult to provide an ideal condition for PDT studies. Inpite of these limitations, in general the articles showedromising results in this field. This review article foundhat for optimal PDT results against cariogenic bacteria, thetructural properties of the bacterium membrane, photo-ensitizer concentration, solubility and polarity, and lightource wavelength must be considered. Specifically for PDTgainst the main cariogenic bacteria (S. mutans), erythrosineems to be the most appropriate photosensitizer since itcts against this Gram positive bacteria, has a hydrophilicendency and even at low concentrations may have photody-amic effects. Considering erythrosin, the most appropriateight source should have a wavelength close to 530 nm, whichay be achieved with the use of low cost LEDs.

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