Comparative evaluation of photosensitizers nitrogen-doped graphene quantum dots and indocyanine green against Enterococcus faecalis - An in vitro microbiological study

Abstract

Background:

The persistent endodontic infections are commonly allied with Enterococcus faecalis, a microorganism capable of forming resistant biofilms that compromise root canal disinfection. Nitrogen-doped graphene quantum dots (N-GQDs) have recently arose as promising photosensitizers for antimicrobial photodynamic therapy (aPDT). The present study equated the antibacterial efficacy of N-GQD-mediated aPDT, indocyanine green (ICG)-mediated aPDT, and diode laser-activated sodium hypochlorite (NaOCl) in contrast to mature E. faecalis biofilms.

Materials and Methods:

Thirty extracted human lower arch premolars were prepared and inoculated with E. faecalis for 14 days. Specimens were arbitrarily allocated to three groups (n = 10): Group I, N-GQDs (100 µg/mL) activated with a 670 nm diode laser; Group II, 0.05% ICG activated with an 810 nm diode laser; and Group III, 3% NaOCl activated with an 810 nm diode laser. Bacterial samples were collected before the treatment and after the treatment, cultured, and quantified by the colony-forming unit (CFU) analysis. Statistical assessments were executed using one-way ANOVA and Bonferroni post hoc tests.

Results:

All the treatment modalities reduced the bacterial counts; however, N-GQD-mediated aPDT demonstrated the greatest antibacterial effect. It showed a 99.27% reduction in CFUs, which was significantly higher than ICG-mediated aPDT (82.05%) and laser-activated NaOCl (62.03%) (P < 0.001).

Conclusion:

N-GQD-mediated aPDT showed superior antibacterial activity against mature E. faecalis biofilms and can serve as an effective adjunctive approach for enhanced root canal disinfection.

INTRODUCTION

Peristent intraradicular infections are an important factor of failure in the root canal therapy. Enterococcus faecalis is the microorganisms usually encountered and it is capable to survive in harsh environmental conditions. Resistance of biofilms to conventional chemomechanical debridement and intracanal antimicrobial agents makes complete eradication challenging.[1,2,3]

Sodium hypochlorite (NaOCl) is known as benchmark irrigant in endodontic practice due to its tissue-dissolving antimicrobial characteristics. Nevertheless, it seems not to be effective enough to bacteria located deep in dentinal tubules or within mature biofilm structures. On the other side, irritation and cytotoxic effects of NaOCl to extruded periapical tissues show the need of adjunctive strategies to augment disinfection.[3,4]

Antimicrobial photodynamic therapy (aPDT) is a supplementary antimicrobial approach consisting on the use of a photosensitizing agent combined by means of light of suitable wavelength to induce reactive oxygen species (ROS), oxidative stress, also inactivation of cellular components of microbial origin.[5] Indocyanine green (ICG) is near-infrared photosensitizer which is approved by Food and Drug Administration and is used in endodontics with demonstrated antimicrobial effects through photodynamic and photothermal mechanisms. However, aggregate and photodegradation might affect its therapeutic efficiency.[6,7]

On the other hand, nitrogen-doped graphene quantum dots (N-GQDs) constitute a new range of carbon-based nanomaterials with appealing optical and electronic characteristics.[8] Nitrogen doping has been proved to improve the photoluminescence and electron-transfer ability of nitrogen-doped N-GQDs (N-N-GQDs), enhancing ROS production.[9] Visible-light diode lasers triggered the potent antimicrobial effects of N-GQDs through combined photodynamic and photothermal actions. The nano-scale dimensions of N-GQDs enable deeper penetration into the dentinal tubules and disruption of established biofilms.[10]

Considering these advantages, N-GQDs may provide enhanced sterile efficacy associated with conventional photosensitizers. Thus, the present study evaluated and compared the antibacterial effectiveness of N-GQD-mediated aPDT, ICG-mediated aPDT, and diode laser–activated NaOCl against mature E. faecalis biofilms within prepared root canals.

MATERIALS AND METHODS

Study design

This was an in vitro experimental study designed to evaluate and compare the antibacterial efficacy of three treatment modalities against E. faecalis biofilms in instrumented root canals. The study protocol was approved by the Institutional Ethical Committee (Approval No: GDCRI/IEC-ACM (2)/14/2024-2025).

Sample preparation

A post hoc power analysis (G*Power 3.1 Heinrich Heine University Düsseldorf in Germany) was performed using effect size derived from pilot data, yielding a power of 0.91 with n = 10 per group, according to which a total of thirty extracted human mandibular premolars were selected [Figure 1a]. All teeth were intact, single-rooted, and free of cracks, caries, or resorption. Extracted teeth were stored in 0.9% Phosphate-buffered saline and used within 1 month to prevent dehydration and microbial contamination. The crowns were removed to standardize the root length at 15 mm. Access cavities were prepared, and canals were shaped using the ProTaper rotary system up to F2 (Dentsply Maillefer, Switzerland).

Figure 1.

(a) Thirty extracted single-rooted human mandibular premolars used as samples for the in vitro study, (b) Aseptic Transfer of culture through paper points, (c) Diode laser irradiation of the root canal using a fiber-optic tip during antimicrobial photodynamic therapy

During instrumentation, canals were irrigated with 5 ml of 3% NaOCl per canal and 3 ml of 17% EDTA for 1 min using 27G side-vented needle placed 1 mm short of working length. Gentle syringe irrigation technique was used. This was followed by a final rinse with 5 ml distilled water for 1 min. All samples were then sterilized by autoclaving to eliminate pre-existing microorganisms.

Bacterial inoculation

A standard strain of E. faecalis (MTCC 9845) was cultured in Brain–Heart Infusion (BHI) broth. Each canal was inoculated with the bacterial suspension and incubated for 14 days at 37°C. Fresh medium was replenished every 48 h to maintain bacterial viability and promote biofilm formation.

Baseline microbial samples were collected using sterile paper points inserted to the working length for 60 s. These samples were transferred to BHI broth for baseline CFU determination [Figure 1b].

Experimental groups

N-GQDs (100 µg/mL) and ICG (0.05%) were procured from Stereorasayan pvt. Ltd Nasik, India.

The samples were randomly divided into three groups (n = 10 each):

1. Group I (Test): N-GQDs (100 µg/mL) + diode laser (670 nm, 0.1 W/cm², 200 µm tip):

   • Preirradiation time: 1 min

   • Irradiation time: 3 min (continuous)

   • Movement: Circular motion within the canal.

2. Group II (Comparator): ICG (0.05%) + diode laser (810 nm, 2.5 W, 200 µm tip):

   • Preirradiation time: 1 min

   • Irradiation time: 2 cycles of 30 s each, with dye reapplication between cycles

   • Movement: Circular motion within the canal.

3. Group III (Control): 3% NaOCl + diode laser (810 nm, 2.5 W, 200 µm tip):

   • No preirradiation time

   • Irradiation time: 2 cycles of 30 s each

   • Movement: Circular motion within the canal.

After each irradiation, canals were flushed with 10 mL of sterile saline and dried with sterile paper points [Figure 1c].

Microbial sampling and colony-forming unit analysis

After treatment, microbial samples were collected using sterile paper points placed at the working length for 60 s. These were transferred into BHI broth, serially diluted, and plated on BHI agar. The plates were incubated under anaerobic conditions at 37°C for 14 days. Colony-forming units (CFU) were counted and expressed as log CFU/mL.

Outcome measure

The primary outcome measure was the logarithmic reduction in E. faecalis CFU after treatment compared to baseline values.

Statistical analysis

Data were analyzed using the statistical package SPSS 26.0 (SPSS Inc., Chicago, IL, USA) and level of significance was set at P < 0.05. Descriptive statistics were performed to assess the mean and standard deviation of respective groups. Normality of the data was assessed by Shapiro–Wilk test. Inferential statistics to find out difference between the group were done by one-way ANOVA test followed by Bonferoni test post hoc test.

RESULTS

Normality assessment

Normality was assessed using the Kolmogorov–Smirnov and Shapiro–Wilk tests. All groups showed P > 0.05, confirming a normal distribution and allowing the use of parametric tests [Table 1].

Table 1.

Normality testing (Kolmogorov–Smirnov and Shapiro–Wilk)

Group	Kolmogorov–Smirnov statistic	Significance	Shapiro–Wilk statistic	Significance
N-GQD	0.217	0.115	0.924	0.117
ICG	0.087	0.200	0.982	0.956
NaOCl	0.156	0.200	0.899	0.089

N-GQD: Nitrogen-doped graphene quantum dots, ICG: Indocyanine green, NaOCl: Sodium hypochlorite

Baseline colony-forming unit counts (before treatment)

Baseline CFU values differed significantly among the N-GQD, ICG, and NaOCl groups.

The NaOCl group had the highest microbial load before treatment, followed by ICG and N-GQ [Table 2].

Table 2.

Baseline colony-forming unit/mL (×10³) with the analysis of variance and post hoc (Bonferroni) comparisons

Group/comparison	n	Mean (×103)	SD	ANOVA F	ANOVA P	Mean difference	Post hoc (P)
N-GQD	10	4.40	0.05	532.65	0.0001*	-	-
ICG	10	4.48	0.06			-	-
NaOCl	10	5.72	0.07			-	-
N-GQD versus ICG	-	-	-	-	-	−0.080	0.0001*
N-GQD versus NaOCl	-	-	-	-	-	−1.320	0.0001*
ICG versus NaOCl	-	-	-	-	-	−1.240	0.0001*

*Significant, N-GQD: Nitrogen-doped graphene quantum dots, ICG: Indocyanine green, NaOCl: Sodium hypochlorite, ANOVA: Analysis of variance, SD: Standard deviation

Posttreatment colony-forming unit counts

There were substantial differences in posttreatment CFU levels.

The N-GQD group demonstrated near-complete elimination, the ICG group showed moderate to strong reduction, and the NaOCl group showed the highest remaining CFU [Table 3].

Table 3.

Posttreatment colony-forming unit/mL (×10³) with analysis of variance and post hoc (Bonferroni) comparisons

Group/comparison	n	Mean (×103)	SD	ANOVA F	ANOVA P	Mean difference	Post hoc (P)
N-GQD	10	0.032	0.039	52.06	0.0001*	-	-
ICG	10	0.804	0.247			-	-
NaOCl	10	2.172	0.784			-	-
N-GQD versus ICG	-	-	-	-	-	−0.772	0.003*
N-GQD versus NaOCl	-	-	-	-	-	−2.140	0.0001*
ICG versus NaOCl	-	-	-	-	-	−1.368	0.0001*

*Significant, N-GQD: Nitrogen-doped graphene quantum dots, ICG: Indocyanine green, NaOCl: Sodium hypochlorite, ANOVA: Analysis of variance, SD: Standard deviation

Percentage reduction in colony-forming unit

Percentage reduction analysis confirmed the superior efficacy of the N-GQD treatment, followed by ICG and NaOCl [Table 4].

Table 4.

Percentage reduction (%), analysis of variance, and post hoc (bonferroni) comparisons

Group/comparison	Mean (%)	SD	Minimum	Maximum	ANOVA F	ANOVA P	Mean difference (%)	Post hoc (P)
N-GQD	99.27	0.89	97.73	100.00	52.06	0.0001*	-	-
ICG	82.05	5.51	75.67	88.84			-	-
NaOCl	62.03	13.70	48.25	80.76			-	-
N-GQD versus ICG	-	-	-	-	-	-	17.22	0.0001*
N-GQD versus NaOCl	-	-	-	-	-	-	37.25	0.0001*
ICG versus NaOCl	-	-	-	-	-	-	20.03

*Significant, N-GQD: Nitrogen-doped graphene quantum dots, ICG: Indocyanine green, NaOCl: Sodium hypochlorite, ANOVA: Analysis of variance, SD: Standard deviation

Figure 2 shows pretreatment and posttreatment bacterial growth plates for the three experimental groups.

Figure 2.

Representative BHI agar plates showing Enterococcus faecalis colony growth before and after treatment in the experimental groups. (a) Group I (N-GQD + diode laser) before treatment, (b) Group I after treatment, (c) Group II (ICG + diode laser) before treatment, (d) Group II after treatment, (e) Group III (NaOCl + diode laser) before treatment, and (f) Group III after treatment

DISCUSSION

Persistent endodontic infections are a significant challenge to the long lasting success of root canal treatment. E. faecalis is one of the main microorganisms involved in these infections. These bacteria are commonly found because it can bear the harsh conditions and survive on limited nutrients. As a Gram-positive facultative anaerobe, it can enter the dentinal tubules to depths extending from about 244 to 1175 µm, making complex biofilm that are more resistant to standard irrigation procedures and intracanal medicines.[1,2,3,4] It is difficult to achieve complete microbial elimination even after careful chemo-mechanical cleaning.

The main irrigating solution which is preferred in endodontics is sodium hypochlorite (NaOCl) because it has antimicrobial effects and can dissolve organic tissues.[3] Although owing to reduced activity when organic material is present and it poor penetration into deeper parts of dentinal tubules, the effectiveness of NaOCl decreases.[4] Therefore, to overcome the limitations of NaOCl, further investigations were carried on additional disinfecting methods that can improve microbial control in the complicated root canal environment.

In endodontic treatment, laser disinfection technique has gained attention as an additional method. Laser irradiation can disturb bacterial cell structures through photothermal and photomechanical interactions, improve microbial reduction in hard-to-reach areas using traditional methods.[11,12] aPDT uses a light-activated photosensitizing agent is also another approach to disinfect the root canal. When activated by the accurate wavelength of light, it produces ROS that damages the microbial cells and can destroy biofilms.[5]

ICG is an infrared photosensitizer that has been widely studied for aPDT applications because of its capacity to produce both the photothermal and the photodynamic antimicrobial effects.[6,7,13] Molecular aggregation and photodegradation may compromise its effectiveness.

Of late, N-GQDs have appeared as the nanomaterials for antimicrobial photodynamic uses. N-GQDs show better photostability, improved electron-transfer properties, and increased ROS production compared to traditional photosensitizers. Their antimicrobial action are enhanced by their small in size which is below 10 nm, it helps them to penetrate dentinal tubules more easily and interact with bacterial biofilms more effectively.[8,9,14] These features are an indication that to target endodontic pathogens, N-GQDs might be a more operative option.

A careful thought is required for the antibacterial potential of N-GQD-mediated aPDT. Treatment time, equipment availability, cost-effectiveness, and procedural ease are the factors that affect its use in clinical environment. In addition, a standard operating procedure and satisfactory training for clinicians are necessary for reliable outcomes. Furthermore, many laboratory, animal trials, and clinical studies are necessary to be carried out to confirm its efficiency and practical value.

The protection in laser-assisted procedures is also of utmost importance. Excessive heat from the laser activation can harm the dentinal and surrounding periodontal tissues, especially if the required procedural instructions and precautions are not taken and not followed. However, there are evidences which show that while delivering effective antimicrobial action, using an optimized laser setting can help to keep temperature rises within safe biological limits.[11,12]

The compatibility of N-GQDs is also another important area of focus. According to the literature and the current studies which are taking place have shown that these nanoparticles have good antimicrobial performance also, they have shown relatively low cytotoxic effects at particular concentrations. Before widespread clinical use can be recommended, dose-dependent responses and uncertainty about long-term biological safety highlights the need for more research.

Group I: Nitrogen-doped graphene quantum dots-mediated antimicrobial photodynamic therapy

The N-GQD-mediated aPDT group showed a mean bacterial reduction of 99.27%, highest antibacterial effectiveness, and undetectable posttreatment CFU counts. This improved antimicrobial action is because of the photodynamic properties from nitrogen doping. Increase in electron-donating capacity, photoluminescence, and increased ROS production after laser exposure is due to the addition of nitrogen to the graphene quantum dot. As stated by Kuo et al., N-GQDs create higher levels of ROS than nondoped graphene quantum dots under similar conditions, leading to better bacterial inactivation and biofilm disruption.[9] Furthermore, the small size of N-GQDs helps them penetrate dentinal tubules, allowing for interaction with bacteria in the areas that are difficult to reach with standard disinfectants. Studies done in the past have shown that graphene quantum dots effectively generate singlet oxygen through multiple sensitization mechanisms, increasing oxidative damage to microbial cells and aiding in biofilm disruption.[14,15] The near-total bacterial eradication achieved in this study corresponds with prior research that highlights the effectiveness of engineered quantum dots as strong photosensitizers for antimicrobial uses.[13,16]

Group II: Indocyanine green-mediated antimicrobial photodynamic therapy

ICG-mediated aPDT led to the substantial decrease in E. faecalis biofilm, with a mean bacterial reduction of 82.05%. While substantial, this reduction was much lower than what was seen with N-GQD-mediated aPDT. When stimulated by near-infrared diode laser irradiation, it causes oxidative damage, disruption of bacterial cell membranes, and protein denaturation, it gives rise to the antimicrobial effect of ICG through photodynamic and photothermal responses.[6,7]

Even after several benefits, there are various factors which can limit the effectiveness of ICG as a photosensitizer. In water, ICG can aggregate and degrade quickly in water, which reduces its ability to produce ROS over time.[13] Early depletion of photosensitizer in the complex root canal environment is the result of inconsistent antimicrobial action and lower rates of bacterial elimination The observation of moderate reduction in CFUs in this study correlates with past laboratory and clinical work done by Leonardo et al., and Higuchi et al. According to them, there was considerable but incomplete bacterial eradication following ICG-based aPDT for endodontic disinfection.[7,13]

Group III: Sodium hypochlorite with diode laser activation

The lowest antibacterial effectiveness among the groups tested, with a mean reduction in bacterial counts of 62.03% were shown by the combination of NaOCl and diode laser irradiation. NaOCl is known for its antimicrobial and tissue-dissolving characteristics, whereas due to inadequate penetration into dentinal tubules and reduced activity when organic debris is present, it’s effectiveness is reduced against mature biofilms and deeply embedded E. faecalis.[4]

The distribution of the irrigant and the stimulation of localized thermal effects is improved by laser activation. However, its capability to generate ROS is limited without a photosensitizer. This explains that the antibacterial impact of laser-activated NaOCl is limited compared to the impact achieved through photodynamic therapy-based methods. The microbial reduction level provided by aPDT is not achieved by laser activation alone as it was stated in the studies which were carried out previously.[15,17]

The N-GQD-mediated aPDT showed highest efficacy, followed by ICG-mediated aPDT and laser-activated NaOCl according to the study. The previous studies demonstrated that nanoparticle-based photodynamic techniques can achieve better microbial elimination than traditional photosensitizers and standard disinfection methods were in consistence with the present study.[15,16,18] However, it was suggested by Chen et al. and Afkhami et al. that factors such as particle size, surface characteristics, and compatibility with the activating light source influence the antimicrobial performance of quantum dots.[18,19]

Limitations

The complex clinical environment of the root canal system, including diverse biofilms, host immune responses, and fluid movement were not completely reflected by this in vitro study. The confounding factors may have been introduced by differences in laser parameters between the experimental groups, which were limiting the direct comparison of treatment outcomes.

Furthermore, the consistency and effectiveness of photodynamic activation were affected, since the laser output was not assessed with a power meter, and the choice of 670 nm as the activation wavelength was not meticulously validated. In this study, only one concentration of N-GQDs and a fixed set of irradiation conditions were analyzed. Antimicrobial effectiveness could have been impacted by the changes in photosensitizer concentration, laser wavelength, exposure time, irradiation methods, or fiber movement.

Furthermore, bias into the results would have been seen because of the differences in baseline CFU values among groups and the absence of an untreated control group. The residual biofilm viability, penetration of antimicrobial agents into dentinal tubules, or potential for bacterial recolonization might not have been fully captured by CFU analysis.

Rather than directly testing the stability and photobleaching behavior of ICG during irradiation, it was inferred from studies conducted in the past. In spite of the promising antibacterial activity, the long-term compatibility, cytotoxicity, and safety of N-GQDs in clinical use need further research.[10,13,18,20]

In spite of these limitations, the findings of this study suggest that N-GQD-mediated aPDT has significant potential as an adjunctive method for eliminating E. faecalis biofilms. Future studies are required which should aim to enhance treatment parameters, evaluate biological safety, and confirm these results in preclinical and clinical settings before considering routine clinical use.

CONCLUSION

The limitations of the current in vitro study, N-GQD-mediated aPDT showed the highest antibacterial effectiveness against E. faecalis biofilms. The N-GQDs had better photodynamic properties, longer generation of ROS, and had the ability to penetrate deeper into dentinal tubules, hence the antimicrobial activity was improved. These features had helped to disrupt mature biofilms in the complicated assembly of the root canal system more effectually. The findings of the present study indicate that for additional disinfection in endodontics, N-GQDs could serve as an effective photosensitizer. However, to fine-tune the treatment conditions, photosensitizer concentration, irradiation methods, and light delivery techniques, there is a need to carry out more research. Further studies should be carried on N-GQDs, to determine how well N-GQDs can penetrate into the tubules, their biocompatibility, and how long does it’s effect lasts. It is the need of the hour to carry out ex vivo studies, animal trials, and clinical studies to evaluate and understand the clinical usefulness, safety, and long-term effectiveness of N-GQD-mediated photodynamic therapy before it can be used in regular endodontic practice.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.