Polymeric Nanoparticle-Based Photodynamic Therapy for Chronic Periodontitis in Vivo

Antimicrobial photodynamic therapy (aPDT) is increasingly being explored for treatment of periodontitis. Here, we investigated the effect of aPDT on human dental plaque bacteria in suspensions and biofilms in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles (MB-NP) and red light at 660 nm. The effect of MB-NP-based aPDT was also evaluated in a clinical pilot study with 10 adult human subjects with chronic periodontitis. Dental plaque samples from human subjects were exposed to aPDT—in planktonic and biofilm phases—with MB or MB-NP (25 µg/mL) at 20 J/cm2 in vitro. Patients were treated either with ultrasonic scaling and scaling and root planing (US + SRP) or ultrasonic scaling + SRP + aPDT with MB-NP (25 µg/mL and 20 J/cm2) in a split-mouth design. In biofilms, MB-NP eliminated approximately 25% more bacteria than free MB. The clinical study demonstrated the safety of aPDT. Both groups showed similar improvements of clinical parameters one month following treatments. However, at three months ultrasonic SRP + aPDT showed a greater effect (28.82%) on gingival bleeding index (GBI) compared to ultrasonic SRP. The utilization of PLGA nanoparticles encapsulated with MB may be a promising adjunct in antimicrobial periodontal treatment.


Introduction
Periodontitis is an inflammatory disease of the supporting tissues of the teeth caused by bacterial infection which, if left untreated, can eventually lead to tooth loss [1]. Tissue destruction occurs as a consequence of the host immune inflammatory response to oral pathogens [2]. Hundreds of different bacterial species have been isolated from periodontal pockets, and a subset of a selected number of species has been associated with periodontitis [3]. Those species participate in the formation of a biofilm on subgingival tooth surfaces in an interdependent form, where early colonizers pose Our hypothesis is that PLGA nanoparticles can improve MB's photodynamic effects and contribute to better clinical outcomes in the treatment of chronic periodontitis. Therefore, in the present study, we investigated the effects of aPDT mediated by MB-loaded PLGA nanoparticles (MB-NP) on human dental plaque microorganisms in vitro (planktonic and biofilm phase) and in vivo (patients with chronic periodontitis).

In Vitro Studies
In both planktonic and biofilm experiments, groups treated only with free MB, MB-NP or light did not show significant differences compared with the control group (no drug/no light), indicating an absence of toxicity for light, MB, or MB-NP alone (data not shown). Table 1 summarizes the effects of aPDT on planktonic and biofilm species following their incubation with free MB or MB-NP. Biofilm bacteria showed greater resistance to aPDT treatment than planktonic cells. MB-MN-mediated aPDT was more effective than free MB-mediated aPDT in both planktonic and biofilm phases. MB-MN-mediated aPDT was equally effective on both planktonic and biofilm microorganisms.
In the planktonic phase, colony forming units (CFU) levels were reduced by 71% and 80% for MB-mediated and MB-NP-mediated aPDT, respectively ( Figure 1). Although MB-NP-mediated aPDT exhibited greater effect on microorganisms in solution compared with free MB, the results were not statistically significant (p > 0.05).
In biofilms, MB-NP-mediated aPDT exhibited 25% greater killing effect compared with free MB ( Figure 2). However, differences between the two groups were not statistically significant (p > 0.05). Our hypothesis is that PLGA nanoparticles can improve MB's photodynamic effects and contribute to better clinical outcomes in the treatment of chronic periodontitis. Therefore, in the present study, we investigated the effects of aPDT mediated by MB-loaded PLGA nanoparticles (MB-NP) on human dental plaque microorganisms in vitro (planktonic and biofilm phase) and in vivo (patients with chronic periodontitis).

In Vitro Studies
In both planktonic and biofilm experiments, groups treated only with free MB, MB-NP or light did not show significant differences compared with the control group (no drug/no light), indicating an absence of toxicity for light, MB, or MB-NP alone (data not shown). Table 1 summarizes the effects of aPDT on planktonic and biofilm species following their incubation with free MB or MB-NP. Biofilm bacteria showed greater resistance to aPDT treatment than planktonic cells. MB-MN-mediated aPDT was more effective than free MB-mediated aPDT in both planktonic and biofilm phases. MB-MN-mediated aPDT was equally effective on both planktonic and biofilm microorganisms.
In the planktonic phase, colony forming units (CFU) levels were reduced by 71% and 80% for MB-mediated and MB-NP-mediated aPDT, respectively ( Figure 1). Although MB-NP-mediated aPDT exhibited greater effect on microorganisms in solution compared with free MB, the results were not statistically significant (p > 0.05).
In biofilms, MB-NP-mediated aPDT exhibited 25% greater killing effect compared with free MB ( Figure 2). However, differences between the two groups were not statistically significant (p > 0.05). Recovered CFU/mL after antimicrobial photodynamic therapy (aPDT) treatment of planktonic bacteria with free methylene blue (MB) (25 µg/mL) and MB-NP (25 µg/mL equivalent to MB) and visible light at 660 nm with an energy fluence of 20 J/cm 2 . Each bar is the mean values of the means from 10 samples (data from each sample were representative of four independent suspensions). Error bars denote the standard deviation of the mean. The asterisks represent the statistical difference between the groups and the control (one-way ANOVA with Tukey's post hoc). *** p < 0.001; ns: not significant; MB: methylene blue; MB-NP: MB-loaded PLGA nanoparticles; CFU: colony forming units.  Figure 1. Recovered CFU/mL after antimicrobial photodynamic therapy (aPDT) treatment of planktonic bacteria with free methylene blue (MB) (25 µg/mL) and MB-NP (25 µg/mL equivalent to MB) and visible light at 660 nm with an energy fluence of 20 J/cm 2 . Each bar is the mean values of the means from 10 samples (data from each sample were representative of four independent suspensions). Error bars denote the standard deviation of the mean. The asterisks represent the statistical difference between the groups and the control (one-way ANOVA with Tukey's post hoc). *** p < 0.001; ns: not significant; MB: methylene blue; MB-NP: MB-loaded PLGA nanoparticles; CFU: colony forming units.

In Vivo Study
After treatments, both groups exhibited a trend of a reduction of moderate and deep sites ( Figure 3). Results were more evident at one month, with a tendency to return to baseline levels by three months after treatments in both groups. Ultrasonic scaling (US) + SRP associated with aPDT had a slightly better outcome than US + SRP alone (p = 0.0298).
Visible plaque index (VPI) was similar for both groups at all time points, with no statistically significant difference observed (Figure 4a; p = 0.9299). After three months, there was a tendency of returning to baseline levels, as noticed for probing pocket depth (PPD). Gingival bleeding index (GBI) percentages decreased drastically and similarly for both groups by one month after treatment (Figure 4b; p = 0.4571). Nonetheless, US + SRP + aPDT had a better performance (28.82%) in preventing GBI compared to US + SRP at three months.

In Vivo Study
After treatments, both groups exhibited a trend of a reduction of moderate and deep sites ( Figure 3). Results were more evident at one month, with a tendency to return to baseline levels by three months after treatments in both groups. Ultrasonic scaling (US) + SRP associated with aPDT had a slightly better outcome than US + SRP alone (p = 0.0298).
Visible plaque index (VPI) was similar for both groups at all time points, with no statistically significant difference observed (Figure 4a; p = 0.9299). After three months, there was a tendency of returning to baseline levels, as noticed for probing pocket depth (PPD). Gingival bleeding index (GBI) percentages decreased drastically and similarly for both groups by one month after treatment ( Figure 4b; p = 0.4571). Nonetheless, US + SRP + aPDT had a better performance (28.82%) in preventing GBI compared to US + SRP at three months.   Overall, except for CAL, all clinical parameters had an improvement at one month for both treatments.    Overall, except for CAL, all clinical parameters had an improvement at one month for both treatments.   The percentage of sites with bleeding on probing (BOP) decreased significantly in both groups one month after treatments, with US + SRP + aPDT group being statistically more effective in reducing BOP than US + SRP (Figure 5a; p = 0.0229). Clinical attachment level (CAL) level was sustained in both groups through all time points with no statistical differences between them (Figure 5b; p = 0.7826). Overall, except for CAL, all clinical parameters had an improvement at one month for both treatments. Overall, except for CAL, all clinical parameters had an improvement at one month for both treatments.

Discussion
Recent meta-analyses on the effect of aPDT for periodontitis showed that the use of aPDT as an adjunct to SRP did not yield better results than SRP alone or associated with systemic antibiotics [38] or provided short-term benefits [39] when administrated as a single session. When applied in multiple sessions, however, aPDT has been proven safe and effective as an adjunctive therapy in periodontal disease treatment, as evidenced by a plethora of studies [10][11][12][13]. Several antimicrobial resistance mechanisms may be responsible for the reduced susceptibility of dental plaque to aPDT. These include the expression of certain phenotypes by biofilm species [40], the slow growing or starved state of microorganisms within biofilms [41], the inactivation of PS [42], the presence of multidrug resistance pumps in bacterial cells that expel the PS [43], and the restricted penetration of PS in oral biofilms [44]. The treatment of biofilm-associated bacterial infections poses challenges due to several antimicrobial resistance mechanisms of biofilms [45]. Possible explanations for the reduced susceptibility of dental plaque to aPDT include one way to overcome the incomplete eradication of dental plaque microorganisms is to develop a delivery system that significantly improves the pharmacological characteristics of the PS.
In the present study, our hypothesis was that MB-loaded PLGA nanoparticles would exhibit a superior photodynamic effect on human dental plaque bacteria-in planktonic and biofilm phase-compared with free MB. Additionally, a pilot study was conducted with 10 patients to evaluate the efficacy of aPDT with MB-NP on chronic periodontitis as an adjunct to ultrasonic scaling and SRP. This is the first in vivo study that employed the use of polymeric nanoparticles as carriers of PS for aPDT. PLGA nanocarriers have been used successfully in drug delivery of MB in vitro, previously [33,46]. MB lacks its photochemical properties when it is encapsulated in PLGA and regains its phototoxicity when it is released by PLGA [47].
In suspensions, the synergism of light and MB-NP showed a greater killing effect (80.5%) over free MB (71%). In oral microcosm laboratory biofilms, nanoparticles and free MB reduced bacterial viability by 79% and 55%, respectively. Although, differences between aPDT groups in both planktonic and biofilm phase were not statistically significant, photodynamic killing results were similar in all experiments. The greater photodynamic effect of MB-loaded nanoparticles over free MB in suspensions and biofilms was also demonstrated by Klepac-Ceraj et al. (2011) [46]. However, in the present study the effect of aPDT on both planktonic and biofilm microorganisms was almost the same; 80.5% vs. 79%, respectively. These data show that nanoparticles were able to penetrate the biofilm and target microorganisms rapidly. Our findings are supported by recent studies that have demonstrated that nanoparticles, regardless their composition, can successfully disrupt the biofilm matrix, allowing for a deeper penetration and a sustained release of drugs [48][49][50][51], as well as increasing drug stability and retention [52][53][54][55].
Our clinical pilot study clearly demonstrated the safety of aPDT. No adverse effects were reported. In this study, the effect of aPDT as an adjunct to US and SRP was compared to US and SRP alone. All clinical parameters (VPI, GBI, BOP, and PPD) in both groups showed the greatest improvement one month following treatment with the exception of CAL that was sustained in both groups through all time points. After one month, all parameters showed a similar increasing trend. At all time points, there were no statistically significant differences between the two treatment groups. However, at three months US + SRP + aPDT showed a greater effect (28.82%) on GBI compared to US + SRP.
At three months after treatment all clinical parameters started to return to baseline levels, for both US + SRP and US + SRP + aPDT, which indicates bacterial recolonization of periodontal pockets. Treatment rebound due to bacterial recolonization is a common feature of chronic periodontitis treatments, regardless the technique employed, as evidenced by studies of Petersilka [56], Zijnge [57], Teles [58], and Sanz-Sánchez [59]. However, the studies of Novaes Jr [60] and Petelin [61] demonstrate that different groups of bacteria are affected after treatment with aPDT or SRP, resulting in a distinct pattern of recolonization. In fact, aPDT was more effective in reducing the presence of Red Complex species, such as Tanerella forsythia and Treponema denticola, and Aggregatibacter actinomycetemcomitans, a species known by its association with localized aggressive periodontitis [60,61]. Taken together, those findings highlight that the association of classical SRP to aPDT in the treatment of periodontitis sums up benefits.
Our findings suggest that MB-NP have the potential to be used as carriers of MB for photodynamic inactivation of dental plaque bacteria. MB-NP and light exhibited a greater killing in biofilms. Our hypothesis is that MB-NP were able to diffuse and released MB within biofilms. This may not be the case in the clinical pilot study that comprised a small number of patients, and, therefore, restricts any broader conclusions. Future studies should define the appropriate aPDT dosimetry (MB concentration, incubation time, power density, and energy fluence) for effective elimination of biofilm species. The possibility of multiple applications of aPDT should also be explored. These improvements and changes in the treatment protocol may demonstrate the adjunctive benefit of aPDT in periodontitis.

Subjects and Samples
Forty-seven patients were analyzed, 27 were excluded due to one or more exclusion criteria, and 20 patients entered the study. Ten patients were assigned to in vitro assays and ten patients participated in the in vivo study. All subjects gave their informed written consent to donate dental samples for inclusion before they participated in the study. The study was conducted at the Dental Office of University of Sao Paulo (Optics Group-Instituto de Fisica de Sao Carlos, Sao Carlos, SP 15980-900, Brazil), and conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Dental School Research Ethics Committee at Araraquara, UNESP Univ. Estadual Paulista (Protocol #04/11). The in vivo study was approved by Human Research Committee-Process HCRP n˝1857/2008. Patients completed a health history questionnaire to ensure that they were medically qualified for participation in the study. Inclusion criteria for the study were patients diagnosed with chronic periodontitis [62] who were no smokers, had at least four teeth in each quadrant (16 teeth on their functional dentition, excluding third molars) and had, at least, two posterior teeth with pocket depth ě4 mm and bleeding on probing (BOP). The exclusion criteria were: smokers, orthodontic brackets, pregnancy, diabetes mellitus, use of anti-inflammatory or antibiotic agents within the previous three months, periodontal therapy during the six months prior to sampling, or use of any medications associated with the gingival disease. The deepest pockets (>5 mm) of each quadrant were used for plaque sampling after the photodynamic and/or periodontal procedure.

In Vitro Study-Sample Collection
Using individual sterile Gracey curettes, dental plaque samples from subgingival sites were taken in each subject (five to eight samples per subject; pockets >5 mm) and placed immediately into pre-reduced, anaerobically sterilized Ringer's solution (Anaerobe Systems, Morgan Hill, CA, USA), forming a sample pool. Bacteria from the plaque samples were dispersed by sonication and homogenezation through Pasteur pipettes. The optical density of the bacterial suspensions was measured in a spectrophotometer and then the pool sample from the ten subjects was divided into two parts, for planktonic and biofilm assays.

Development of Plaque-Derived Biofilms
For biofilm development, the bacterial inoculum (in BHI broth) was adjusted to contain approximately 10 7 cells/mL. Approximately 1.5ˆ10 6 bacteria (150 µL) were dispensed into a blood agar well. For each experimental group 4 wells were used. Plates were incubated at 35˝C in anaerobic atmosphere (80% N 2 , 10% H 2 , and 10% CO 2 ) for seven days. At day 2 the broth was carefully aspirated and fresh BHI broth was added to each well. Then, fresh BHI broth was added daily to each well, very slowly, to avoid disruption of the biofilm.

Photodynamic Treatment in Vitro
A diode laser with a central wavelength of 660 nm coupled to a 1 mm optical fiber that delivered light into a lens was used for both planktonic and biofilm studies. The system formed a uniform circular spot, 2 cm in diameter, which was able to irradiate a group of four wells in a 96-well plate each time, from above, at room temperature in the absence of surrounding light. The power density was measured using a powermeter. For both planktonic and biofilm experiments power density was 100 mW/cm 2 and energy fluence was 20 J/cm 2 . Microorganisms in planktonic and biofilm phase were exposed only once to light. Free MB concentration was 25 µg/mL and the final concentration of MB-NP was 25 µg/mL equivalent to MB. Experimental groups were: (1)  Aliquots of bacterial suspensions (10 8 cells/mL) were placed in sterile microtubes and centrifuged at 7000 rpm for 4 min. One milliliter of sterile free MB or MB-NP was then added after discarding the supernatants. Bacterial cells were suspended in free MB or MB-NP and placed in four wells of 96-well plates for 10 min before exposure to light. Following aPDT, bacterial suspensions underwent serial dilutions in BHI broth, and 100 µL aliquots were plated on blood agar and incubated under anaerobic conditions at 35˝C for seven days prior to CFU scoring.

Biofilms
Carefully, growth medium was aspirated from each well of 96-well plates and replaced by 150 µL of sterile free MB or MB-NP. Biofilms were then incubated for 10 min followed by exposure to light. After aPDT, bacteria from each well were gently scraped using a sterile bacteriological loop, dispersed in BHI broth and measured in a spectrophotometer at 600 nm. After, serial dilutions were prepared and 100 µL aliquots were plated on blood agar plates, which were incubated anaerobically at 35˝C for seven days prior to CFU counting.

Preparation and Characterization of PLGA Nanocarriers
MB loaded into PLGA nanoparticles (10% w/w) were prepared in the Department of Pharmaceutical Sciences at Northeastern University as previously described [33]. Briefly, a PLGA (76 mg) and Pluronic ® F-108 (14 mg) solution was prepared in 5 mL of acetone. MB as oleate salt (Sigma Chemicals Co.) was dissolved at 10% (w/w) concentration in the PLGA acetone solution for the preparation of the MB-loaded nanoparticles. To insure that the formed nanoparticles have a stable hydrophilic surface, which resists aggregation, pluronic triblock copolymers were added to the polymer solution in acetone at 20% (w/w). The acetone solution was added into an aqueous (50 mL) solution under vigorous stirring and left to stir overnight. The following day, nanoparticles were centrifuged at 10,000 rpm for 20 min, washed twice with deionized distilled water and lyophilized. Data regarding nanoparticle characterization were previously published [33]. Figure 6 shows a scanning electron micrograph of blank PLGA nanoparticles.

In Vivo Study
This 3-month study evaluated clinically the effectiveness of the adjunctive use of polymeric nanoparticle-based aPDT following periodontal instrumentation with ultrasonic scaling and mechanical scaling and root planing (SRP) in patients (seven men; 13 women; aged 20-70) with moderate to advanced chronic periodontitis [62]. All enroled patients completed the study. The study investigated the correlation of the clinical parameters before and after aPDT treatment in periodontitis sites in the same patient, following a split-mouth design. All four quadrants received treatment. Two of them (one lower and one upper jaw) received non-PDT ultrasonic scaling followed by mechanical SRP with Gracey curettes and the other two quadrants received ultrasonic scaling (US) and mechanical SRP followed by aPDT. Prior to aPDT, MB-NP were applied as a mouthwash (MB-NP dispersed in PBS 1×) and then periodontal pockets were irrigated with the same PS solution for 10 min. PDT was applied as a single session. The effect of the two different treatment groups-US + SRP vs. US + SRP + aPDT-was investigated on clinical parameters such as probing pocket depth (PPD), visible plaque index (VPI), gingival bleeding index (GBI), bleeding on probing (BOP), and clinical attachment level (CAL). All clinical parameters measured at baseline, one week, one month, and three months, and recorded by a single examiner. Oral hygiene procedures were instructed and reinforced at every appointment. PPD at baseline was divided into two categories: shallow sites (pocket depth from 1 to 3 mm) and moderate to deep sites (moderate: 4-6 mm; deep ≥ 7 mm).

Data Collection-Measurement Reproducibility
Calibration trials were performed prior to the study to ensure adequate intra-examiner reproducibility (kappa statistic ≥ 90%). Intra-examiner kappa values were 0.97 (PPD) and 0.93 (CAL). All measurements were performed by a single examiner using a standard University of North Carolina probe with millimeter markings.

Clinical Parameters
Clinical parameters that were examined in this study included presence or absence of visible plaque index (VPI), gingival bleeding index (GBI), and bleeding on probing (BOP). Full-mouth probing pocket depth (PPD) and clinical attachment level (CAL) were measured by a North Carolina manual periodontal probe (North Caroline Probe, Hu-Friedy, Chicago, IL, USA) at six sites per tooth in all teeth except third molars, at baseline, one week, one month, and three months after the aPDT and non-aPDT associated with ultrasonic scaling and periodontal treatment (US-SRP).

In Vivo Study
This 3-month study evaluated clinically the effectiveness of the adjunctive use of polymeric nanoparticle-based aPDT following periodontal instrumentation with ultrasonic scaling and mechanical scaling and root planing (SRP) in patients (seven men; 13 women; aged 20-70) with moderate to advanced chronic periodontitis [62]. All enroled patients completed the study. The study investigated the correlation of the clinical parameters before and after aPDT treatment in periodontitis sites in the same patient, following a split-mouth design. All four quadrants received treatment. Two of them (one lower and one upper jaw) received non-PDT ultrasonic scaling followed by mechanical SRP with Gracey curettes and the other two quadrants received ultrasonic scaling (US) and mechanical SRP followed by aPDT. Prior to aPDT, MB-NP were applied as a mouthwash (MB-NP dispersed in PBS 1ˆ) and then periodontal pockets were irrigated with the same PS solution for 10 min. PDT was applied as a single session. The effect of the two different treatment groups-US + SRP vs. US + SRP + aPDT-was investigated on clinical parameters such as probing pocket depth (PPD), visible plaque index (VPI), gingival bleeding index (GBI), bleeding on probing (BOP), and clinical attachment level (CAL). All clinical parameters measured at baseline, one week, one month, and three months, and recorded by a single examiner. Oral hygiene procedures were instructed and reinforced at every appointment. PPD at baseline was divided into two categories: shallow sites (pocket depth from 1 to 3 mm) and moderate to deep sites (moderate: 4-6 mm; deep ě 7 mm).

Data Collection-Measurement Reproducibility
Calibration trials were performed prior to the study to ensure adequate intra-examiner reproducibility (kappa statistic ě 90%). Intra-examiner kappa values were 0.97 (PPD) and 0.93 (CAL). All measurements were performed by a single examiner using a standard University of North Carolina probe with millimeter markings.

Clinical Parameters
Clinical parameters that were examined in this study included presence or absence of visible plaque index (VPI), gingival bleeding index (GBI), and bleeding on probing (BOP). Full-mouth probing pocket depth (PPD) and clinical attachment level (CAL) were measured by a North Carolina manual periodontal probe (North Caroline Probe, Hu-Friedy, Chicago, IL, USA) at six sites per tooth in all teeth except third molars, at baseline, one week, one month, and three months after the aPDT and non-aPDT associated with ultrasonic scaling and periodontal treatment (US-SRP).

Statistical Analysis
In vitro data were expressed as the mean plus standard deviation (SD) and were analyzed by one-way ANOVA with Tukey's post hoc test using GraphPad Prism ® Version 5.01 software (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered to be significant when p < 0.05 (confidence level of 95%). For in vivo data, differences between groups were sought using the repeated measures t-test, also using GraphPad Prism ® Version 5.01 software. Differences with a p-value <0.05 at a confidence level of 95% were considered significant.