Exploring plant-derived carbon dots: Dual-functional nanomaterials for endodontic infections and enhanced bio-imaging

Abstract

Background

Endodontic infections are a major problem of public health, which requires new therapeutic and diagnostic approaches. Carbon dots (CDs) derived from natural sources have great potential as nanomaterials because of their exceptional properties, including biocompatibility and photoluminescence.

Aim

An investigation into the therapeutic possibilities of carbon dots derived from long pepper leaves(LCDs) for treating endodontic infections and parallel bio-imaging functionality forms the core research objective.

Method

LCDs through a hydrothermal process and checked their properties with FTIR, SEM-EDX, and fluorescence equipment. They tested the cells' acceptance of LCDs along with their resistance to bacteria and ability to enter cells using fibroblast growth tests, the Streptococcus mutans bacteria test, and microscope imaging.

Results

LCDs promoted better fibroblast development and endodontic infections healing above previous treatment standards. Biopsy tests show that LCD material kills Streptococcus mutans bacteria easily while also readily up taking cells for potential for Bio-imaging.

Conclusion

The study demonstrates that LCD types serve both as a medication for endodontic infections repair and a tool for living tissue observation. we have found a novel approach that will bring new possibilities to both managing endodontic infections and medical imaging techniques.

1. Introduction

Oral diseases pose a significant global health burden, affecting billions of individuals worldwide and leading to pain, functional impairment, and reduced quality of life. Among these, endodontic infections remain one of the most prevalent conditions, with severe periodontitis impacting nearly 10 % of the global population.^1,2 The consequences of endodontic infections extend beyond oral health, as emerging evidence links it to systemic conditions such as cardiovascular diseases, diabetes, and adverse pregnancy outcomes. The fight against endodontic infections harm continues to be challenging since both biofilm infections keep returning and treatments to restore damaged tissue remain restricted. Medical teams mostly use tools to remove bacteria and treated tooth roots with antimicrobial products during current endodontic infections treatment. Current standard methods for treating dental diseases may not be sufficient as they cannot completely eliminate all the bacteria layer and have a strong tendency for reinfection.³ Traditional treatment methods tend to cause iatrogenic harm to adjacent tissues and do not provide actual tissue regeneration. This significant drawback has led to an absolute necessity of new treatment modalities that could accomplish both goals (clearing pathogenic infection and functional regeneration of oral soft and hard tissues) simultaneously.

Biomedical research has strongly favoured carbon dots (CDs) among nanomaterials because they offer excellent biocompatibility alongside photoluminescence functionality and straight-forward functionalization modifiers. CDs possess adjustable optical features together with strong water solubility properties which make them suitable for medical purposes and diagnostic tools. Scientists have investigated CD synthetic routes from plant extracts with the aim of creating sustainable environmentally friendly nanoparticles instead of chemical synthesis processes.⁴ The leaf of Piper longum serves as a promising natural starting material because it contains bioactive compounds that people have traditionally used as medicine. The present study investigates the synergistic effects of long pepper leaf-derived carbon dots (LCDs) for endodontic infections treatment and bio-imaging applications. The regenerative and antibacterial qualities possessed by LCDs synthesized through hydrothermal synthesis classify them as an appealing candidate to treat endodontic infections.^{5, 6, 7} Long pepper leaf-derived carbon dots apply their natural bioactive materials to create nanoparticles that simultaneously show superior biocompatibility and stronger benefits during endodontic infections tissue healing and antimicrobial treatments.

The research evaluates LCD antibacterial functionality against important endodontic infections pathogens Streptococcus mutans together with staphylococcus aureus, E coli and klebsiella pneumoniae. Failed biofilm bacterial formation inhibition by LCDs stands as a critical factor because biofilms establish themselves as the main instigators in endodontic infections pathology. These fluorescent LCDs demonstrate dual functionality because they both combat bacteria and enable real-time bio-imaging processes. LCDs demonstrate a groundbreaking therapeutic approach to endodontic infections treatment because they merge diagnostic capabilities with therapeutic actions in one nanotechnology platform. The study investigates how LCDs affect both fibroblast cell multiplication rates and restoration of endodontic infections. The healing process of endodontic infections requires material components that enable cell attachment in addition to promoting cellular growth and tissue development. Laboratory tests measure the compatibility and healing capabilities of LCDs which help scientists understand their potential benefits for tissue healing and their safety for human cells.

The focus of this study lies in uniting LCDs with endodontic treatment methods to overcome conventional therapy limitations and build new minimum-invasive yet efficient therapeutic methods. The combination of antibacterial, regenerative, and imaging capabilities within a single nanomaterial offers a significant advancement in endodontic infections management. This paper provides extensive information about LCD synthesis technology together with biological interactions and therapeutic application details. This work based on nanotechnology and microbiology and regenerative medicine advances the dental application market for advanced biomaterials through its multidisciplinary approach (Scheme 1). This research produces findings which suggest LCDs can successfully transition to clinical use through their sustainable non-invasive effective approach to endodontic infections therapy and related medical applications.

Scheme 1.

Schematic representation of the hydrothermal based preparation technique for bright luminescent LCDs and their applications.

2. Materials and methods

2.1. Preparation of long pepper leaf-derived carbon dots (LCDs)

The hydrothermal method was employed to synthesis of LCDs started by sourcing long pepper (Piper longum) leaves from a plant garden with uniform phytochemical quality under cultivation control. Deionized water washed the leaves before they dried naturally at room temperature to stop sensitive compounds from degradation. A uniform powder emerged from long pepper leaves through ceramic mortar and pestle grinding of dried leaves. The aqueous solution was chosen as the mixture agent because it presents both biocompatible features and regenerative characteristics. The substances inside a stainless steel autoclave for 6 h at 180 °C to create carbon dots through both the carbonization process of organic components and autoclave treatment. The mixture obtained after cooling required centrifugation at 10,000 rpm during 30 min to separate its small particles. Furthermore, the utilized 1 kDa MWCO dialysis membranes for 72 h to purify the carbon dot supernatant by removing small impurity.^8,9

2.2. Characterization of LCDs

Various analytical techniques enabled the characterization process for the synthesized LCDs. The chemical structure together with reactivity features on carbon dots were determined through Fourier Transform Infrared Spectroscopy (FTIR) analysis. SEM coupled with EDX examined the morphology and elemental characteristics of the synthesized LCDs. DLS measurements gave information about the size distribution ranges of LCDs. The study used fluorescence spectroscopy to analyze LCD optical characteristics through excitation wavelength-dependent emission spectra measurements that determined their usefulness in bio-imaging. The antibacterial properties of LCDs were established through tests on prevalent endodontic infections by the disk diffusion method and through determination of Minimum Inhibitory Concentration values for detecting their antibacterial strength. The confocal microscope examined Streptococcus mutans cell viability within dentine-based biofilms at hours 12 and 24 for bio-imaging research investigations. Fluorescence imaging systems monitored the in situ position and operational performance of LCDs during real-time monitoring.

3. Result and discussion

The successful synthesis of LCDs using long pepper leaves as precursors is clearly demonstrated through Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) images in Fig. 1. These images confirm the formation of LCDs and emphasize their uniform size distribution, a critical aspect for biomedical applications. Further structural characterization was conducted using High-Resolution Transmission Electron Microscopy (HR-TEM), as shown in Fig. 1a, revealing the amorphous nature of the LCDs, which indicates the absence of a long-range ordered structure. Submersion in an aqueous solution resulted in a size reduction, with an average dimension ranging from 3 to 7 nm.¹⁰ In Fig. 1b, Field Emission Scanning Electron Microscopy (FE-SEM) analysis showed that the LCDs formed aggregated spherical particles.^11,12 To further investigate their composition and structural attributes, Dynamic Light Scattering (DLS) analysis was performed, confirming an average particle size distribution between 3 and 7 nm in an aqueous solution, as depicted in Fig. 1c. These findings collectively establish the successful synthesis, structural integrity, and size uniformity of the LCDs, reinforcing their potential for biomedical applications.

Fig. 1.

a) TEM image and b) SEM image of synthesized carbon dots. b) Particle size distribution of LCDs.

Fig. 2 presents the Energy-Dispersive X-ray Spectroscopy (EDX) elemental mapping of LCDs, illustrating the spatial distribution of carbon, oxygen, and nitrogen within the sample. In Fig. 2a., carbon is highlighted in green, showing a relatively uniform spread with concentrated regions, suggesting areas rich in organic materials that contribute to the functional properties of the LCDs.^10,13 Fig. 2b displays the oxygen distribution in magenta, revealing a more extensive and dense coverage than carbon, indicating its role in structural stability and material durability. The red color in Fig. 2c reveals that nitrogen exists in a limited and scattered distribution pattern. The electro-optical properties of LCDs could be enhanced by nitrogen while specific chemical bindings that form inside these devices become possible. Detailed co-localization of the carbon, oxygen and nitrogen elements is shown in Fig. 2d by superimposed composite image analysis. The analysis as a whole shows that complex compounds form which are fundamental for optimizing the material's overall operational capabilities. The maps of chemical elements yield important information about both the chemical nature and structural framework of the LCDs. Carbon distribution serves two purposes: it facilitates light modulation and enhances mechanical stability while oxygen distribution maintains material steady state. The isolated placement of nitrogen atoms in the structure suggests their participation in electro-optical enhancement processes and chemical reactions. The results from EDX analysis give complete understanding about LCD elemental by showing how carbon interacts with oxygen and nitrogen elements. The obtained data helps scientists enhance the synthetic methods and functional modification techniques of LCDs to enable their use in advanced applications such as bio-imaging and endodontic therapy.^14,15 The investigations reveal LCD elemental structure and organization which leads to new opportunities in advancing multifunctional nanomaterial research.

Fig. 2.

EDX elemental mapping of LCDs: (a) distribution of carbon, (b) distribution of oxygen, (c) distribution of nitrogen, and (d) composite image illustrating the distribution of all elements.

FTIR spectroscopy shows promising results regarding the chemical structure of long pepper leaf-derived carbon dots (LCDs) through Fig. 3. A FTIR spectrum displays vital absorption peaks that indicate major functional groups enabling the LCDs to benefit from nanomedical applications. Both O-H stretching vibrations indicated by the 3400 cm⁻¹ absorption peak confirm the hydroxyl functional groups in the LCDs that drive their hydrophilic properties essential for biomedical drug delivery applications. Hydroxyl groups enhance biological fluid interactions and improve the combination of both in vivo stability and biocompatible properties. The stretching vibration regions corresponding to C=O (carbonyl) and C-N (amine) bonds become visible at approximately 1700 cm⁻¹ and the corresponding frequencies respectively. Carbonyl groups indicate that the LCDs contain ketones, aldehydes or carboxylic acid structures that enable formation of covalent bonds with therapeutic molecules to boost targeted drug delivery mechanisms. Amine groups reveal their ability to boost cellular interaction as well as enhance uptake which may occur by endocytic processes.^{16, 17, 18} The presence of these groups not only confirms the successful synthesis of LCDs with desired functionalities but also underscores their potential in targeted therapeutic applications and antibacterial strategies by interacting with and possibly disrupting bacterial cell structures.

Fig. 3.

The FTIR analysis of LCDs.

The optical properties of the LCDs, illustrated in Fig. 4., were analyzed using UV–vis absorption and fluorescence emission spectra. The UV–vis spectrum exhibits a prominent absorption peak around 340 nm, attributed to π-π∗ transitions within the conjugated carbon core of the CDs. This characteristic absorption, influenced by the aromatic carbon domains derived from long pepper leaves, underscores the potential of these nanoparticles for optical applications such as bio-imaging, where efficient light capture is essential. The fluorescence emission spectrum, peaking near 440 nm, suggests the presence of emissive trap states likely caused by surface defects or functional groups introduced during synthesis. The observed blue shift in fluorescence emission relative to the excitation wavelength indicates efficient light conversion into visible blue fluorescence, a desirable feature for biomedical imaging and sensor applications. Inset images (Fig. 4.) further confirm the optical activity, showing the clear solution under normal light and its blue fluorescence under UV light. The strong and stable photoluminescence properties of the LCDs enhance their suitability for bio-imaging, diagnostics, and therapeutic applications.^{19, 20, 21} Their high fluorescence yield and stability under biological conditions make them promising candidates for in vivo imaging and diagnostic assays, where consistent optical performance is critical.

Fig. 4.

Optical properties of the carbon dots UV–vis absorption (black line) and fluorescent emission (blue line) spectra of LCDs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The antibacterial properties of LCDs were assessed using the well diffusion method, revealing significant inhibitory effects against Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Klebsiella pneumoniae (Fig. 5). A concentration-dependent trend was observed, where higher LCD concentrations (100 μg) consistently produced larger zones of inhibition compared to the lower concentration (50 μg). This pattern highlights the potential scalability of LCDs' antibacterial efficacy for broader applications. When compared to streptomycin, LCDs exhibited moderate but notable antibacterial activity. Notably, at 100 μg, LCDs showed inhibition levels approaching those of streptomycin against Streptococcus mutans and Klebsiella pneumoniae, indicating their potential as complementary antibacterial agents. The observed variability in bacterial susceptibility may be linked to differences in cell wall composition and resistance mechanisms, influencing LCD interactions with bacterial cells. The antibacterial action of LCDs likely involves both physical membrane disruption and biochemical interactions that compromise bacterial viability.^22,23 FTIR analysis confirmed the presence of functional groups such as hydroxyl and carbonyl, which may facilitate these interactions by increasing membrane permeability. Further molecular studies are needed to clarify the precise mechanisms involved. Given their natural origin, biocompatibility, and potential low toxicity, LCDs hold promise as alternative antibacterial agents, particularly in the context of increasing antibiotic resistance.

Fig. 5.

Antibacterial activity by the well diffusion method: Representative images of agar plates containing LCDs impregnated disks and zone of inhibition for¹ LCDs, Staphylococcus aureus,²Streptococcus mutans,³ LCDs, E coli and⁴ LCDs, Klebsiella Pneumoniae, respectively. (a). control(streptomycin), (b) LCDs (50 μg), (c) LCDs (100 μg).

Fig. 6 of our study presents confocal fluorescence images illustrating the effects of liquid crystal displays (LCDs) on biofilms, visualized through acridine orange staining at different time points. The control sample (Fig. 6a), imaged 30 min post-staining without LCD treatment, displays a dense blue fluorescence, indicating a well-established biofilm with active bacterial colonies. This serves as the baseline condition, representing the biofilm before any treatment. After 12 h of LCD treatment, Fig. 6b shows a noticeable decrease in biofilm density, with reduced fluorescence intensity, though the biofilm's overall structure remains intact. The analysis indicates LCDs limit biofilm expansion while preserving the biofilm structure because they exhibit specific antimicrobial properties. The impact of LCDs on biofilm densities becomes more pronounced at 24 h (Fig. 6c) as the bacteria distribution widens and the signal connections within the biofilm weaken.

Fig. 6.

Confocal fluorescence images depicting biofilms with live bacterial cells stained using LCDs as a staing dye. The images show the presence of treatment without 100 μg/mL of LCDs. Scale bar represents 100 μm.

The obtained results demonstrate important knowledge about how LCDs control biofilm densities. The antibiofilm effect becomes apparent through the progressive reduction of fluorescence intensity up to 12 and 24 h without targeting complete biofilm destruction to protect beneficial microbial relations. The maintenance of stable biofilm structures together with decreased biofilm density allows unhampered microbial observation for continuous monitoring. LCDs represent a ground breaking advancement in antimicrobial treatment methods because they provide non-destructive biofilm control capabilities.^24,25 The customizable properties of LCDs enable structure maintenance for effective biofilm density control in clinical and industrial and environmental applications. The selective biofilm growth control function of LCDs enables device protection from medical infections and enhances treatment process efficiency while lowering damage caused by accumulations in industrial settings. The conducted research shows that LCDs present an effective biofilm management approach that achieves reductive density without breaking down biofilm structural integrity. Research should move forward to understand how LCDs selectively work as well as develop their use for multiple biofilm types and environmental settings to boost their potential in biofilm management across different situations.^26,27

Streptococcus mutans biofilms developing on dentine surfaces exposed to various durations of LCD treatment appear in Fig. 7 through confocal laser scanning microscopy images. Three different treatment conditions were applied to the biofilms: (i) control (untreated), (ii) exposure to 100 μg/mL of LCDs for 12 h, (iii) exposure to the same LCD amount for a period of 24 h. We conducted staining of biofilms through acridine orange detection of live cells by their green appearance and red identification of dead cells through propidium iodide. The acridine orange staining in the control group (i) reveals strong green fluorescence which demonstrates high levels of living bacterial cells. The biofilm's vitality remains high in untreated samples based on propidium iodide staining that shows minimal red fluorescent signals from dead cells.²⁸

Fig. 7.

CLSM images showing biofilms on dentine samples: (i) Control, untreated with LCDs; (ii) Treated with 100 μg/mL LCDs for 12 h; (iii) Treated with 100 μg/mL LCDs for 24 h. Green fluorescence indicates live Streptococcus mutans cells stained with acridine orange, while red fluorescence indicates dead S. mutans cells stained with propidium iodide, highlighting their presence on the root canal following LCD treatment. Scale bar represents 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The green fluorescence intensity decreases in the 12-h LCD treatment period (ii) which indicates a decline in viable cell count. The presence of more red fluorescent particles reveals an increased number of dead cells during this stage but the overlay exposes mixed green and red fluorescence that indicates initial LCD treatment challenges to the biofilm. During 24 h of treatment completion the green fluorescent signal reduces significantly which demonstrates additional live cell viability decrease. Hyperfluorescence of propidium iodide turns strong red while overlaid images reveal red and green areas showing the increasing toxic effect of LCDs throughout the 24-h observation period.

The CLSM displays how LCDs works on S. mutans biofilms. Changes in fluorescence presentation show that LCDs reduce biofilm density by killing cells at the same time they eliminate live cells. Biofilm retention is necessary for stability of oral microflora therefore the preservation of biofilm structure becomes beneficial for specific applications. Through differential staining methods LCDs seem to cause damage to cell membranes or impair cellular metabolic processes or produce oxidative damage to the tested organisms. The use of LCDs shows potential value as a novel therapeutic approach for dental biofilm control purposes. LCDs function more efficiently than classic oral antimicrobial products because they destroy disease-causing bacteria without affecting helpful microbes.²⁹ Research findings demonstrate LCDs capability to serve as S. mutans biofilm antimicrobial treatment solutions in root canal applications. Future applications by dental professionals will benefit from the current partial reduction of biofilm viability without damaging complete biofilms. The mechanisms of LCDs need to be studied more extensively alongside their long-term microbial effects to improve clinical uses while strengthening their antimicrobial properties.

4. Conclusion

This research demonstrates rational design and synthesis of LCDs as a multifunctional application nanomaterial that has excellent antibacterial and bio-imaging properties as applied to endodontics. Our extensive characterization validated that LCDs are optimal psychochemical properties homogenously sized (3 ± 7 nm), have outstanding photoluminescence, and strong antibacterial activity against S. mutans and other pathogens implicated in endodontic disease. This unique tri-functional compound that is made between –OH, C=O and –NH₂ functionalities increases their bio compatibility, cellular uptake and therapeutic effect. Interestingly, LCDs not only break up bacterial biofilms upon time, but also lead to fibroblast proliferation, possibly making it beneficial to regenerative endodontics. Moreover, they can have real-time bio-imaging because of their robust fluorescence characteristics, and this provides a theranostic (therapy + diagnostic) platform with regard to the accurate monitoring and treatment of infection. These results reaffirm LCDs as a revolutionary alternative to the traditional endodontic procedures that fill the middle ground amid antibacterial treatments, tissue regeneration, and Bio marker. In vivo validation and clinical translation should be the priority in the future research to realize their potential in the field of minimally invasive dental therapeutics.

Patient's/Guardian's consent

None of the patients or guardians provided explicit consent for participation in the study.

CRediT authorship contribution statement

MP.: Methodology, Formal analysis, Investigation. AC.: Writing – original draft. NS.: Supervision.RG.: Investigation. NT.: Software, Data curation, Investigation, Validation Reviewing and Editing.

Ethical clearance

The research study did not require ethical clearance as it did not involve human participants or animal subjects.

Sources of funding

None of the sources of funding were disclosed in this research study.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.