ORIGINAL ARTICLE

Safety assessment of oral photodynamic therapy in rats

Carla R. Fontana & Mark A. Lerman & Niraj Patel &Clovis Grecco & Carlos A. de Souza Costa &

Mansoor M. Amiji & Vanderlei S. Bagnato &

Nikolaos S. Soukos

Received: 27 January 2012 /Accepted: 22 March 2012 /Published online: 31 March 2012# Springer-Verlag London Ltd 2012

Abstract Photodynamic therapy (PDT) is based on thesynergism of a photosensitive drug (a photosensitizer)and visible light to destroy target cells (e.g., malignant,premalignant, or bacterial cells). The aim of this studywas to investigate the response of normal rat tonguemucosa to PDT following the topical application of he-matoporphyrin derivative (Photogem®), Photodithazine®,methylene blue (MB), and poly(lactic-co-glycolic acid)

(PLGA) nanoparticles loaded with MB. One hundred andthirty three rats were randomly divided in variousgroups: the PDT groups were treated with the photo-sensitizers for 10 min followed by exposure to red light.Those in control groups received neither photosensitizernor light, and they were subjected to light exposure aloneor to photosensitizer alone. Fluorescent signals wereobtained from tongue tissue immediately after the topicalapplication of photosensitizers and 24 h following PDT.Histological changes were evaluated at baseline and at 1,3, 7, and 15 days post-PDT treatment. Fluorescence wasdetected immediately after the application of the photosen-sitizers, but not 24 h following PDT. Histology revealedintact mucosa in all experimental groups at all evaluationtime points. The results suggest that there is a therapeuticwindow where PDT with Photogem®, Photodithazine®,MB, and MB-loaded PLGA nanoparticles could safelytarget oral pathogenic bacteria without damaging normaloral tissue.

Keywords Photodynamic therapy . Photosensitizers .

Histological analysis . Fluorescence

Introduction

Photodynamic therapy (PDT) was developed as a therapyfor cancer and is based on the concept that a nontoxicphotosensitizing agent, known as photosensitizer, can bepreferentially localized in premalignant and malignant tis-sues and subsequently activated by light of the appropriatewavelength to generate singlet oxygen and free radicalsthat are cytotoxic to cells of the target tissue [1]. PDT hasfound its greatest success as a treatment for cancer, age-related macular degeneration [2], actinic keratosis [3], and

C. R. Fontana (*)Department of Clinical Analysis, School of PharmaceuticalSciences, University of São Paulo State (UNESP),1621 Expedicionarios do Brasil Street,Araraquara, Sau Paulo 14801-960, Brazile-mail: fontanacr@fcfar.unesp.br

M. A. LermanDepartment of Oral Medicine, Infection and Immunity,Harvard School of Dental Medicine,Boston, MA 02115, USA

N. Patel :N. S. SoukosApplied Molecular Photomedicine Laboratory,The Forsyth Institute,Boston, MA 02115, USA

C. Grecco :V. S. BagnatoInstitute of Physics of São Carlos, University of São Paulo (USP),400 Trabalhador São Carlense Avenue,São Carlos, Sau Paulo 15980-900, Brazil

C. A. de Souza CostaDental School of Araraquara, University of São Paulo State (UNESP),Araraquara, Brazil

N. Patel :M. M. AmijiDepartment of Pharmaceutical Sciences, School of Pharmacy,Bouvé College of Health Sciences, Northeastern University,Boston, MA 02115, USA

Lasers Med Sci (2013) 28:479–486DOI 10.1007/s10103-012-1091-6

Barrett's esophagus [4]. The application of PDT for target-ing pathogenic microbes in wound infections has beenexplored in animal models [5–7]. PDT with topical appli-cation of 5-aminolevulinic acid is used off-label for treat-ment of acne vulgaris [8] and has been employed forclinical use as an antifungal agent [9]. In the dental field,PDT was approved for palliative treatment of patients withadvanced head and neck cancer in the European Union,Norway, and Iceland.

Most of the photosensitizers under investigation forcancer treatment are based on the tetrapyrrole nucleus,such as porphyrins, chlorins, bacteriochlorins, and phtha-locyanines [10]. Photogem®, a hematoporphyrin deriva-tive similar to Photofrin II, and Photodithazine®, achlorin e6 derivative, were used for targeting cancer inhuman [11–13] and animal [14, 15] studies recently. Onthe other hand, antimicrobial photosensitizers such as por-phyrins, phthalocyanines, and phenothiazines (toluidineblue O, methylene blue) can directly target both gram-negative and gram-positive bacteria [16]. Methylene blue(MB), whose intravenous administration is FDA-approvedfor methemoglobinemia, bears a positive charge and hasbeen used for targeting oral bacteria in PDT studies [17].The product called Periowave is a laser system with apatient treatment kit of methylene blue that was commer-cialized by Ondine Biopharma Corporation in Canada forthe treatment of periodontitis. We have explored a newapproach for antimicrobial therapy with light activationof targeted MB-loaded poly(lactic-co-glycolic acid)(PLGA) nanoparticles [18]. Once encapsulated withinPLGA, the excited state of the photosensitizer is quenched,which results in loss of phototoxicity [19]. When the nano-particles are incubated with the targeted cells, they show atime-dependent release of the photosensitizer, which thenregains its phototoxicity and results in an activatable PDTnanoagent [20].

PDT as a local treatment of oral infection (dental caries,periodontal diseases, peri-implantitis, endodontic therapy,moniliasis), either in combination with traditional methodsor by itself, arises as a simple, nontoxic, and inexpensivemodality. However, PDT effectiveness has not been con-firmed due to lack of adequate evidence of safety andefficacy as well as lack of optimization of dosimetry. Studieshave been performed using different treatment conditionsand parameters with insufficient clinical findings. The ob-jective of this study was to assess the photodynamic effectsof Photogem®, Photodithazine®, MB, and MB-loadedPLGA nanoparticles on the normal tongue in rats. Ourhypothesis was that topical application of the photosensi-tizers on the tongue dorsum followed by exposure to visiblelight, with drug and light parameters similar to those thatmay be applied in a clinical setting, would not induce anycytotoxic effects.

Materials and methods

Animals

One hundred and thirty three male Wistar rats (350–400 g)were obtained from the animal facilities at the MedicalSchool of Ribeirão Preto–University of São Paulo. The ratswere housed in individual cages and were fed standard ratpellets and tap water ad libitum. The study design andanimal experimental procedures were reviewed and ap-proved by the Research Ethics Committee, University ofSão Paulo at Ribeirão Preto Medical School. Before alloperative procedures, the animals were anesthetized byan intramuscular injection of ketamine hydrochloride(Ketamina 10 mL; Virbac do Brasil Ind. Com. Ltda.,Roseira, SP, Brazil) at a dose of 0.10 mL/100 g bodyweight, associated with a muscular relaxant and analgesic(xylazine; Schering-Plough Saude Animal Ind Com.Ltda, Cotia, SP, Brazil) at a dose of 0.05 mL/100 g bodyweight. The rats were immobilized at a homemade sur-gical table, allowing a reasonable mouth opening to carryout the tongue photosensitization and illumination.

Photosensitizers

Photogem® Stock solution of Photogem® (Limited LiabilityCompany Photogem, Moscow, Russia), a first-generationhematoporphyrin-derived photosensitizer whose clinical usehas been approved by the Brazilian National Health Surveil-lance Agency (ANVISA), was prepared at pH06.6 by dis-solving the powder in sterile phosphate buffered saline (PBS)and kept in the dark. The concentration of 30 mg/mL wasused in this study. The ultraviolet-visible absorption spectraof Photogem® are characterized by a long wavelength, max-imum at 630 nm [21].

Photodithazine® Stock solution of Photodithazine® (Rada-Farma, Moscow, Russia) was prepared in sterile PBS andkept in the dark. The concentration of 5 mg/mL was used inthis study.

Methylene blue Methylene blue (Sigma) was dissolved insterile PBS to give a solution at concentration of 50 μg/mLbefore use.

PLGA nanocarriers (a) Preparation: PLGA nanoparticlesencapsulating MB (10 % w/w) were prepared by blendingthe medical-grade PLGA (MW 12 kDa, 50:50 lactide/gly-colide molar ratio; Birmingham Polymers, Pelham, AL)with Pluronic® F-108 triblock copolymer (PerformanceChemicals Division of BASF, Parsipanny, NJ), and fabricat-ing the nanoparticles by a solvent displacement procedure[22]. Briefly, a solution of PLGA (76 mg) and Pluronic®

480 Lasers Med Sci (2013) 28:479–486

F-108 (14 mg) was prepared in acetone (5 mL) and heatedwith stirring until it became clear. For the preparation of theMB-loaded nanoparticles, the oleate salt of MB (SigmaChemicals Co.) was dissolved at 10 % (w/w) concentrationin the acetone solution of PLGA. Pluronic® triblock copoly-mers were added to the polymer solution in acetone at 20 %(w/w) to insure that the formed nanocarriers have a stablehydrophilic surface, which resists aggregation. The solutionwas introduced into an aqueous (50 mL) solution undervigorous stirring and left to stir overnight. The next day,nanoparticles were centrifuged at 10,000 rpm for 20 min,then washed twice with deionized distilled water and lyoph-ilized under vacuum for 48 h. To modify the surface prop-erties of nanoparticles with anionic charge, we used cetyltrimethyl ammonium bromide (Sigma Chemicals Co.) as asurfactant. (b) Characterization: The mean size of PLGAnanoparticles, with and without the encapsulated payloads,was determined via laser light scattering using a ZetaPALSsystem (Brookhaven Instruments, Holtsville, NY). The sur-face morphology of the nanocarriers was visualized byscanning electron microscopy (Shimadzu, Japan) followingfreeze-drying. The surface charge on the nanoparticles, inthe presence and absence of encapsulated payload, wasdetermined by zeta potential measurements of the nanocar-rier suspensions in PBS (pH 7.4) with ZetaPALS (phaseanalysis light scattering) ultra-sensitive zeta potential ana-lyzer. To determine the amount of drug loaded into thenanocarriers (capacity) as well as the percentage of addeddrug (efficiency), a known amount (~10 mg) of the controland poly(ethylene oxide) (PEO)-modified nanocarriers wasdissolved in acetone. The amount of encapsulated drug inthe nanocarriers was determined by using the UV-vis absor-bance of MB. The release kinetics of MB-oleate salt fromthe nanoparticles was determined in PBS (pH 7.4). Toincrease the solubility of MB complex, Tween1-80, a non-ionic surfactant, was added to the release medium at 1.0 %(w/v) concentration. This also prevented the MB-loadednanocarriers from binding to the container surface. Onehundred milligrams of the drug-containing nanocarrierswas incubated with 10 mL of the release medium in ashaking water bath (50 rpm). Periodically, 5 mL of therelease medium was removed and replaced with 5 mL offresh buffer to maintain sink conditions. MB in the releasemedium was assayed by Shimadzu UV-Vis spectrophotom-eter (Columbia, MD). Cumulative amount and percent drugreleased was determined from appropriate calibration curvesof the respective agents.

Photodynamic therapy

Animals were randomly assigned to the following 11groups: [1] no light/no photosensitizer (control group, 9

animals); [2] light at 630 nm only (12 animals); [3] light at660 nm only (12 animals); [4] Photogem® only (12 ani-mals); [5] Photodithazine® only (12 animals); [6] MB only(12 animals); [7] MB-loaded PLGA nanoparticles only (12animals); [8] Photogem® and light at 630 nm (13 animals);[9] Photodithazine® and light at 660 nm (13 animals); [10]MB and light at 660 nm (13 animals); and [11] MB-loadedPLGA nanoparticles and light at 660 (13 animals). In group1, sterile saline was applied on the dorsum of the tongue for10 min. In groups 4, 5, 6, and 7, the photosensitizer wasapplied on the dorsum of the tongue for 10 min using asterilized swab (a micropipette; Boeco, Hamburg, Germany).In groups 8–11, light was applied for 10 min following theapplication of the photosensitizer for 10 min.

Two light emitting diode (LED) systems (LED Edixeon®;Edison Opto Corporation, New Taipei City, Taiwan) with anoutput power of 120 mW were used to deliver light withcentral wavelengths at 630 nm (groups 2 and 8) and 660 nm(groups 3, 9, 10, and 11). The illuminated tongue area wasdetermined by the spot light placed in contact with the tonguedorsum, 0.5 cm away from the apex of the tongue (Fig. 1). Thespot area was 1 cm2 for both wavelengths. The power densi-ties of incident radiation were measured with a power meter(Coherent-FM-LM10) and were found to be 153 mW/cm2 for630 nm and 120 mW/cm2 for 660 nm. The energy fluenciesfor 630 and 660 nm were 91.8 and 72 J/cm2, respectively.

Clinical evaluation

Animals were anesthetized, and macroscopic changes in thetongue mucosa were evaluated at baseline and at 1, 3, 7, and15 days posttreatment by a single calibrated examiner usinga four-point scoring system: 0, completely intact tonguemucosa; 1, slight redness without discontinuation of tissue;2, severe damage with tissue erosion; and 3, carbonization

Fig. 1 The area on the dorsum exposed to light was 0.5 cm away fromthe apex of the tongue

Lasers Med Sci (2013) 28:479–486 481

or ulcer formation reaching more than 50 % of surface area.Standardized photographs of the tongue were obtained witha digital camera (Nikon SLR camera D40x, 10.2 megapixel,Tokyo, Japan).

Fluorescence spectroscopy

Fluorescence spectra were obtained with a probe placed onthe tongue at baseline and 24 h following PDT. Each spec-trum was taken in less than 10 s using a system composed ofa doubled Nd:YAG laser emitting at 532 nm and a spec-trometer that collected fluorescence in the range of 350–850 nm. The excitation laser was coupled to a Y type probe,which was connected at one end to the laser and to thespectrometer at the other. A 110-μm central fiber deliveredthe excitation light, and six surrounding ones, 100 μm each,collected the reemitted light from the target tissue; the totaldiameter of the investigation tip is around 2 mm. Thecollected light was filtered with a band pass filter, minimiz-ing the scattered light. The fluorescence spectrum wasobtained with the probe perpendicularly placed in gentlecontact to the tongue.

Histological evaluation

Following fluorescence measurements, rats were sacri-ficed using anesthetic overdose with an intraperitonealinjection of ketamine hydrochloride associated with amuscular relaxant and analgesic xylazine hydrochlorideat baseline and at 1, 3, 7, and 15 days posttreatment(Table 1). The whole tongue was removed in a singlepiece. For histological analysis, the samples were imme-diately immersed in 10 % formalin for 4 days at roomtemperature and subjected to routine processing. Serial6-μm thick histological sections were obtained with arotary microtome (820 Spencer Microtome, Spencer

Products Co., Carson, CA, USA), stained with hematox-ylin and eosin, and observed with a Carl Zeiss lightmicroscope. The slides were examined by a pathologistblinded to the experimental and control groups. A de-scriptive analysis of the histological characteristics of thetissue was performed using the criteria presented inTable 2. As an indicator of orthokeratosis, the thicknessof keratin was measured and expressed as a percentageof the total thickness of epithelium measured from thebasal layer through the superficial keratin.

Results

Characterization of nanoparticles

PLGA nanoparticles were spherical in shape and had asmooth surface [23]. UV-visible spectroscopy verified thecapacity and efficiency of MB encapsulation. The averagediameter of PEO-PLGA nanoparticles was approximately200 nm. The particle size remained the same with theinclusion of MB. The surface charge of the nanocarriers,in the absence and presence of encapsulated payload, wasdetermined by zeta potential measurements and was foundto be −23.5 and−31.9 mV, respectively. These averagevalues were obtained from six independent batches of nano-particles and were not statistically significant (p>0.05). UV-visible spectroscopy verified the capacity and efficiency ofMB encapsulation. [23].

Clinical assessment

The rat tongue mucosa remained intact and similar to thecontrol group (no light/no photosensitizer) in all experimen-tal groups and at all evaluation time points. No erythemawas observed in any of the treated animals.

Table 1 Number of rats sacri-ficed per group at baseline(0 h) and at 1, 3, 7, and15 days following PDT

Animal groups 0 h 24 h 72 h 7 days 15 days Total

Control (no light/no drug) 3 3 1 1 1 9

Only light (630 nm) 3 3 2 2 2 12

Only light (660 nm) 3 3 2 2 2 12

Only Photogem® 3 3 2 2 2 12

Only Photodithazine® 3 3 2 2 2 12

Only methylene blue 3 3 2 2 2 12

Only nanoparticles 3 3 2 2 2 12

Photogem® + light (630 nm) 3 3 3 2 2 13

Photodithazine® + light (660 nm) 3 3 3 2 2 13

Methylene Blue + light (660 nm) 3 3 3 2 2 13

Nanoparticles + light (660 nm) 3 3 3 2 2 13

133

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Fluorescence detection

Fluorescence spectra were obtained from tongue tissue im-mediately after the topical application of photosensitizersand 24 h following PDT (Fig. 2). Following application ofthe photosensitizers for 10 min, the appearance of the emis-sion bands associated with the presence of the agents wasevident in all spectra. Photogem® showed two characteristicemission bands at 618 and 680 nm as previously shown[24]. Photodithazine®, MB, and MB-loaded PLGA nano-particles showed a single emission band at 660–670 nm.Twenty-four hours following photosensitizer applicationand exposure to light, all agents were cleared from thetongue tissue, and typical autofluorescence spectra were

obtained that were identical to those obtained from thetongue mucosa prior to photosensitizer application.

Histological assessment

Histology revealed intact tongue mucosa in all experimentalgroups at all time points. The rat tongue epithelium in group 1(no light/no photosensitizer; control group) was stratified withuniform layers (Fig. 3a). An average of 10–15 distinct epithe-lial layers was counted, and an average 40–45 % orthoker-atosis was estimated. Neither ulceration nor significant mitoticactivity was observed in the epithelium. The underlying con-nective tissue showed numerous regional striated muscle bun-dles with normal vascularity and a minimal inflammatory cellinfiltrate. No distinct histological differences were detectedbetween the control tongue epithelium and the other groups(only light, only photosensitizer, PDT) for the parametersstudied (Table 2). Neither group exhibited necrosis of epithe-lium or a strong inflammatory infiltrate underlying the intactepithelium (Fig. 3b–f).

Discussion

The role of photodynamic therapy as a local treatment of oralinfection, either in combination with traditional methods oforal care or alone, arises as a simple and inexpensive modalitywith little risk of microbial resistance [25]. There are severalfactors influencing photodamage, including the type, dose,incubation time and localization of the photosensitizer, the

Table 2 Histological criteria used to evaluate the effect of photosensi-tizers, light, and PDT on the rat tongue mucosa

Histological criteria

Epithelium

Average number of distinct epithelial cell layers

Average % orthokeratosis

% area ulceration

Average mitoses/high-power field

Connective tissue

Normal/loose/dense

Thickness

Inflammatory cell infiltrate

Vascularity

Fig. 2 Fluorescence spectraobtained from the tonguedorsum of rats immediately and24 h following topicalapplication of thephotosensitizers. For the plotobtained immediately after theapplication of the compound,the autofluorescence wassubtracted from the tissuespectra

Lasers Med Sci (2013) 28:479–486 483

availability of oxygen, the wavelength of light (in nano-meters), the light power density (in megawatts per squarecentimeter), and the light energy fluence (in Joules per squarecentimeter). In the present study, we assessed the photody-namic effects ofMB,MB-loaded PLGA nanoparticles, Photo-gem®, and Photodithazine® on the normal tongue of rats. Nocytotoxic effects were observed on the tongue dorsum, sug-gesting that PDT with drug and light parameters similar tothose that may be applied for antibacterial targeting in aclinical setting displays a safe therapeutic window.

MB has been used as a photosensitizing agent since the1920s [26]. The application of MB-mediated PDT in clinicalstudies using either the Periowave™ Treatment kit or theHelbo® Blue treatment kit, at concentrations of 50 μg/mLand 10 mg/mL, respectively, is as follows: MB is applieddirectly in the dental pockets for 60 s followed by exposureto red light via a fiberoptic probe for 60 s per pocket or pertooth (10 s per site, six sites in total). In the majority of thesestudies, PDT as an adjunct to scaling and root planing did

not show any beneficial effects over scaling and root planingalone [25]. It is possible that short exposures to light may beresponsible for the lack of clinical benefits. In the presentstudy, the concentration of MB was 50 μg/mL, and itsincubation time was longer (10 min). In addition, the powerdensities and energy fluences used in our study were muchgreater, 120–153 mW/cm2 and 72–92 J/cm2, respectively,compared with those used in clinical studies. This suggeststhat there is still plenty of safe space for increasing the lightparameters in these studies. Recently, the potential of PDTwas suggested as an adjunctive technique to eliminate re-sidual root canal bacteria following standard endodonticchemo-mechanical debridement. The safety of MB-mediatedPDT for endodontic disinfection has been addressed in tworecent in vitro studies [27, 28]. Concentrations of MB rangingfrom 37 to 370 μg/mL produced up to 36 % killing forfibroblasts after incubation for 20 min followed by exposureto red light with a total energy of 36 J [27]. The viability ofhuman gingival fibroblasts and osteoblasts was studied in

Fig. 3 Sagittal sections of thetongue dorsum in rats. In thecontrol group (no light/nophotosensitizer), theorthokeratinized stratifiedsquamous epithelium overliesthe fibrous connective tissuethat contains numerous regionalstriated muscle bundles (a).Immediately following PDTwith Photogem® (b),Photodithazine® (c), MB (d),and MB-loaded nanoparticles(e), scattered chronicinflammatory cells wereobserved in the connectivetissue underneath the intactepithelium. Twenty-four hoursfollowing PDT with Photodi-thazine® (f), no changes wereobserved in the tongueepithelium and connectivetissue. Hematoxylin–eosin, ×400

484 Lasers Med Sci (2013) 28:479–486

vitro after exposure to MB and red light with parameterssimilar to those that may be applied in a clinical setting. Bothcell types were sensitized with 50 μg/mL of MB followed byexposure to red light at 665 nm for 5 min with an irradiance of10, 20, and 40 mW⁄cm2. Light at 40 mW⁄cm2 with MB hadmodest effects on cells. The clinical use of MB for PDT ofbladder [29] and esophageal [30] cancers and its use in photo-targeting Helicobacter pylori in the rat gastric mucosa [31],along with the data obtained in this study, suggest that thelocal use of MB is safe.

The use of PLGA nanoparticles as carriers of photosensi-tizers has been recently explored in antimicrobial photody-namic therapy [19, 23]. The nanoagents offer a concentratedpackage of photosensitizer for the production of reactiveoxygen species that destroy cells, limit the ability of micro-organisms to pump the photosensitizer molecule back out,and that can have selectivity of treatment by either passivetargeting or by active targeting via the charged surface of thenanoparticle [25]. MB-loaded PLGA nanoparticles, with aconcentration of 50 mg/mL equivalent to MB, exhibited agreater PDT killing compared with free MB on microorgan-isms in dental plaque samples obtained from patients withchronic periodontitis as well as on microcosm biofilmsoriginated directly from the whole-mixed natural plaque[23]. Our findings here suggest that MB-loaded PLGAnanoparticles can be safely applied topically for photodes-truction of oral bacteria. This field of research is very young,and many parameters should be determined in future stud-ies, such as the amount of MB encapsulated in nanopar-ticles, the physical characteristics of nanoparticles (e.g.,size, zeta potential) that are important in determining theirintracellular uptake and trafficking, the incubation time ofMB-loaded nanoparticles with bacteria, and the optimalPDT parameters for effective elimination of biofilm micro-organisms. However, the use of biodegradable polymer tosynthesize the nanoparticles makes the final product attrac-tive for clinical use. The nanoparticle matrix PLGA is apolyester copolymer of polylactide and polyglycolide thathas received approval by the US Food and Drug Adminis-tration as a result of its biocompatibility and its ability todegrade in the body through natural pathways [32].

Photogem®, a hematoporphyrin derivative similar toPhotofrin II, and Photodithazine®, a glucosamine salt ofchlorin e6, are photosensitizers developed in Russia andwere used in antimicrobial PDT previously. Photogem®was used for targeting Streptococcus mutans biofilms invitro [33] as well as different Candida species and othermicroorganism on dentures in vitro [34, 35]. Recently, Pho-togem® was used for treatment of denture stomatitis causedby Candida species in hunan subjects with dentures [36].The concentration of Photogem® used for eradication of S.mutans biofilms was 0.25 mg/mL followed by exposure tolight with energy fluence up to 150 J/cm2 [33]. In our study,

we used 120-fold greater concentration of Photogem® and92 J/cm2 of red light at 630 nm. In an in vivo study, the hardpalate of patients with denture stomatitis was sprayed with500 mg/mL Photogem® followed by exposure to light at455 nm 30 min later with energy fluence of 122 J/cm2 [36],suggesting that much greater doses of Photogem® could beapplied compared to that used in our study. On the otherhand, Photodithazine® was used for the inactivation ofCandida guilliermondii in vitro [37]. Here, we used 5 mg/mL Photodithazine® topically, whereas Wen et al. [38]injected 10 mg of Photodithazine® per kilogram of bodyweight in mice.

Microbial biofilms in the oral cavity are involved in theetiology of various oral conditions, including caries, peri-odontal and endodontic diseases, candidiasis, denture sto-matitis, oral malodor, and dental implant failures. Theresults of the present study suggest that there is a therapeuticwindow where PDT with Photogem®, MB, Photoditha-zine®, and MB-loaded PLGA nanoparticles could targetoral biofilm bacteria safely and rapidly without damagingnormal tissue.

Acknowledgments This work was supported by FAPESP.

Disclosure of proprietary interests We certify that we have noaffiliation with or financial involvement in any organization or entitywith a direct financial interest in the subject matter or materials discussedin the manuscript (e.g., employment, consultancies, stock ownership,honoraria).

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