In vitro evaluation of antimicrobial and cytotoxic activity of newly synthesized and characterized nanocurcumin

Abstract

Purpose:

Curcumin, the principal curcuminoid found in turmeric, demonstrates potent antibacterial and anti-inflammatory characteristics; nevertheless, it possesses restricted solubility and bioavailability. Numerous studies have reported elevated bioavailability and antimicrobial activity of nano-formulations of curcumin. Porphyromonas gingivalis plays a crucial role in triggering dysbiosis in the subgingival milieu, leading to the progression of periodontitis. This work details nanocurcumin preparation, characterization, and in vitro evaluation of antibacterial activity against Porphyromonas gingivalis and its cytotoxic effects on L929 mouse embryo fibroblasts.

Materials and methods:

Nanocurcumin was prepared using the antisolvent precipitation method and characterized by Scanning electron microscopy, UV-visible spectrophotometry, Fourier transform infrared spectroscopy, and Nuclear Magnetic Resonance. The antimicrobial activity of nanocurcumin against Porphyromonas gingivalis was assessed using the minimum inhibitory concentration (MIC). Cytotoxicity against L929 mouse embryo fibroblasts was evaluated with the MTT assay.

Results:

Nanocurcumin exhibited significant water solubility, nanoparticle size ranging from 18 to 176 nm, a strong absorbance peak at 425nm, and a comparable chemical structure to curcumin. MIC of nanocurcumin was 6.25 µg/ml compared to 25 µg/ml of curcumin and 3.12 µg/ml of tetracycline. Both nanocurcumin and curcumin demonstrated a dose-dependent negative impact on L929 fibroblasts with IC50 concentrations of 86.16 µg/ml and 45.69 µg/ml respectively.

Conclusion:

Nanocurcumin in the present study demonstrates well-characterized nanoparticle formation with excellent antimicrobial activity against Porphyromonas gingivalis and minimal cytotoxicity against L929 cell line. Nanocurcumin demonstrates significant promise for integration into drug delivery systems for the management of periodontal disease, subject to additional experimental and clinical trials

Introduction

Curcumin(C) is derived from the rhizome of Curcuma longa and is a natural polyphenolic compound. C, demethoxycurcumin, and bisdemethoxycurcumin are collectively known as curcuminoids (1). C is a commonly used ingredient in food and has demonstrated therapeutic potential in rheumatism, liver disorders, respiratory diseases, and wound healing. It has anti-inflammatory, anti-cancer, anti-bacterial, anti-fungal, and antioxidant properties (2). Multiple studies have reported the antibacterial effects of C against Porphyromonas gingivalis (P. gingivalis), Prevotella intermedia, Fusobacterium nucleatum, Treponema denticola and other periodontal pathogens (3). Despite multiple health benefits, C demonstrates low solubility and limited bioavailability in vivo (4). Possible causes include low absorption, chemical instability, rapid breakdown, and swift systemic clearance (5).

Nanocurcumin (NC) is water soluble and demonstrates high in vivo bioavailability with a 60-fold rise in biological half-life compared to bulk C (6), resulting in an increased ability to eliminate microbes causing intracellular infections (7). Numerous investigations have documented the cytotoxic effects of NC on cancer cell lines (8, 9) and antimicrobial activity against periodontal pathogens. The antimicrobial efficacy of curcumin-loaded silica nanoparticles has been assessed against clinically isolated P. gingivalis (10), and the activity of curcumin nanocrystals against P. gingivalis from implant failure has also been documented (11). Oral NC is recognised for its anti-inflammatory properties by inhibiting Interleukin-1β, Interleukin-6, and Tumour Necrosis Factor-α, in patients with COVID-19 (12). Locally administered curcumin-loaded nanoparticles inhibited inflammation and bone resorption in rats with experimentally induced periodontal disease (13). Thus, NC demonstrates potential for host modulation.

Periodontitis is an inflammatory disease that damages the soft tissues surrounding the tooth and is the major cause of tooth loss in adults. The initiating factor of periodontitis is microorganisms colonizing the periodontal pockets and P. gingivalis is considered the keystone pathogen of periodontitis (14). Since P. gingivalis can enter gingival tissues, mechanical debridement is almost ineffective, and periodontal pockets may get recolonized with P. gingivalis resulting in recurrence of periodontitis (15). The systemic administration of antimicrobial agents can lead to the development of antibiotic-resistant strains and inability to achieve adequate gingival sulcular fluid concentration (16). Subgingival local drug delivery (LDD) can overcome the systemic side effects and drug concentration after application is significantly more than the minimum inhibitory concentration which can persist for up to several weeks (17). Although antibiotics are commonly used as LDD agents, the risk of developing antimicrobial resistance precludes their use. Consequently, numerous phytotherapeutic compounds are being examined for their potential as subgingival local drug delivery agents.

To the best of our knowledge, the antibacterial efficacy of pure NC against laboratory strains of P. gingivalis and its cytotoxic effects on healthy fibroblasts have not been assessed. This study intends to prepare and characterize NC, and to assess its in vitro antibacterial efficacy against P. gingivalis and cytotoxicity towards L929 mouse embryo cells.

Materials and methods

Ethical approval

This study was granted approval by the Institutional Ethics Committee (PMS/IEC/2022/REGULAR/APR/34).

Sources of materials

Synthetic C with purity of 97.0% was obtained from TCI Chemicals, Japan. L929- Mouse embryo fibroblast cell line was procured from the National Centre for Cell Science, Pune, India. P. gingivalis ATCC strain was purchased from ATCC, Virginia, USA. Chloroform, dimethyl sulfoxide, and phosphate buffer saline were purchased from Sigma Aldrich, Missouri, USA. Dulbecco's modified eagle's medium, MTT reagent, and Mueller Hinton broth were purchased from Himedia, Thane, India. Tryptic soy broth was procured from Gibco, Massachusetts, USA. All chemicals used in this study were of analytical grade.

Preparation of nanocurcumin

NC was synthesized utilizing the antisolvent precipitation technique previously outlined, with minor modifications (18). Briefly, 10mg of C was dissolved in 20 ml of chloroform and introduced dropwise to boiling water at 100°C under magnetic stirring for two hours. After the supernatant was removed, the solution was subjected to centrifugation at 8000 rpm for 15 minutes. The residue is subsequently dried in a hot air oven at 50°C to yield NC particles.

Characterization of nanocurcumin

The UV-visible absorption spectra of NC and C dissolved in Dimethyl sulfoxide (DMSO) were measured with a Bio spectrometer (Eppendorf fluorescence, Hamburg, Germany) between the range 250 to 600 nm. A scanning Electron Microscope (SEM) was utilized to observe surface morphology analysis including shape, roughness, and aggregation. The morphology of NC was assessed using a Field Emission Scanning Electron Microscope (Zeiss SIGMA 360 VP, Oberkochen, Germany) equipped with Bruker EDX at 5 kV accelerating voltage. C morphology was assessed with a Scanning Electron Microscope (Jeol 6390LA/ OXFORD XMX N, Tokyo, Japan) at 20kV accelerating voltage. The size of nanoparticles was evaluated using basic Image J software. Fourier transform infrared spectroscopy (FTIR) was evaluated with Nicolet iS50 Fourier Transform Infrared Absorption Spectra (FTIR) system (Thermo Fisher Scientific, Massachusetts, USA) to analyze the chemical structure of C and NC. The measurements were performed at a resolution of 4 cm-1 over the wavenumber range of 4000 cm-1 to 100 cm-1. The Proton Nuclear Magnetic Resonance (1H NMR) spectrum of NC was taken using Bruker Avance III HD 400 MHz spectrometer (Bruker Corporation, Massachusetts, USA) by dissolving NC in deuterated chloroform (CDCl3) as the solvent.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

P. gingivalis strain ATCC 33277 was maintained in blood agar plates in anaerobic conditions. The colonies isolated were diluted in fresh Tryptic soy broth (Gibco, USA) supplemented with Vitamin K (0.1%) and hemin (0.5%) to achieve the turbidity of 0.5 McFarland standards. For further experiments, the aliquots of this bacterial suspension were utilized. Broth microdilution method using serial dilutions was used to determine MIC according to the method reported by the Clinical and Laboratory Standards Institute (19). C and NC were dissolved in dimethyl sulfoxide to obtain 200 μg/ml. Dilutions of the sample were prepared with a twofold serial dilution method in concentrations ranging from 100 to 0.05 μg/ml in Mueller Hinton broth. Tetracycline was used as positive control and broth solution was the negative growth control. The wells were inoculated with 100μl P. gingivalis strain and incubated under anaerobic conditions for 48 hours at 37°C. The antibacterial action of NC after 48 hours was determined visually by detecting a lack of turbidity indicating bacterial growth inhibition. The lowest concentration with no turbidity was considered the MIC. The assay was performed in triplicate. To determine Minimum Bactericidal Concentration (MBC), 100 µl of aliquots demonstrating the absence of bacterial growth during MIC evaluation were spread on blood agar plates and incubated anaerobically at 37 °C for 48h followed by bacterial growth analysis.

Cytotoxicity

Cytotoxicity was determined on the L929- Mouse embryo fibroblast cell line with MTT assay. Cells were cultured in Dulbecco's modified eagle's medium- a high glucose medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution, in a CO2 incubator at 37°C. Subculturing was performed every 2 days. Cell passage number 04 was used for the current study. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) method is based on the formazan purple crystal formation rate, which is assessed in terms of absorbance at 570 nm and is directly proportional to the number of live cells. The following formula was used to calculate the percentage of cell viability:Percentage cell viability=((Mean absorbance of treated cells)/(Mean absorbance of untreated cells))*100. The cell viability of NC was assessed at concentrations ranging from 3.125 and 400 µg/ml. C at an equivalent concentration served as the positive control, whereas untreated L929 cells functioned as the negative control.

Statistical analysis

Intergroup and intragroup comparison of C and NC cell viability at various concentrations were analyzed using unpaired t-test and repeated measure ANOVA with Tukey’s HSD post hoc test respectively using statistical software Statistical Package for Social Sciences, Version 22.0 (IBM, Armonk, NY, United States). The level of significance was fixed at p-value less than 0.05. Linear regression equation Y =Mx+C. Y = 50 was used to determine IC50 value. Cell viability graph was used to derive values of M and C.

Results

Water solubility

When suspended in deionized water, the NC powder dispersed rapidly and seemed soluble, but C was insoluble in water, exhibiting visible particles (Figure 1, Inset).

Figure 1.

Group 1 (Control) treated samples before and after aging, respectively, 3D images, from top to bottom Cerasmart (A-D), Vita Enamic (B-E), InitUV-Vis spectra of Curcumin (a) and Nanocurcumin (b) in dimethyl sulfoxide. Inset: Solutions of (c) Nanocurcumin and (d) Curcumin in deionized water.

Characterization results

UV Vis Spectrophotometry revealed strong absorption peaks at 425nm for NC and 435nm for C (Figure 1). SEM examination indicated that bulk C had an uneven morphology with particle sizes varying from 1.4 to 33 µm (Figure 2a). The shape of NC particles was mostly spherical, with few flower-like nanostructures ranging in particle size from 18 to 176 nm. (Figure 2b). FTIR spectrum of NC revealed major peaks at 3503,3270, 3014, 2919, 2850, and 1729 cm-1, compared to peaks at 3507, 3274, 2916 and 2848 cm-1 for C. A small additional peak at 1729 cm-1 indicates the presence of an aldehyde group in NC, which is absent in C spectra. Changes noted in the absorption peaks between the two compounds were minor (Figure 3). The 1H NMR spectrum of the protons of NC is depicted in Figure 4a with well-separated peaks. The chemical shifts (δ) are assigned to individual C molecule protons. The chemical structure of C with labelled carbon atoms is depicted in Figure 4b (20). The prominent peak at δ 3.951 ppm shows the presence of the methoxy group. The singlet at δ 5.80 reveals the presence of an intact olefinic proton. The peaks between δ 6.75 to 7.75 ppm reveal the presence of aromatic protons. These data reveal the identical chemical structures of NC and C.

Figure 2.

Scanning electron microscopic image of (a) Curcumin and (b) Nanocurcumin

Figure 3.

Fourier transform infrared spectroscopy of (a) Curcumin and (b) Nanocurcumin

Figure 4.

(a) Proton nuclear magnetic resonance (1H NMR) spectrum of curcumin in deuterated chloroform (b) Structure of curcumin

MIC and MBC

The MIC values for tetracycline, C, and NC were 3.12 µg/ml, 25 µg/ml, and 6.25 µg/ml, respectively (Figure 5a). Each compound's MBC values were 6.25µg/ml for tetracycline, 50µg/ml for C, and 12.5µg/ml for NC (Figure 5b).

Figure 5.

(a) Minimum Inhibitory Concentration (MIC) by broth microdilution method. A. Tetracycline B. Curcumin C. Nanocurcumin (b) Minimum Bactericidal Concentration (MBC) of Curcumin, Nanocurcumin and Tetracycline

Cytotoxicity

MTT assay revealed that with the increase of NC and C concentrations from 3.125µg to 400µg in L929 cells, cell viability diminished from 97.67% to 13.77% for NC and from 95.24% to 5.12% for C (Figure 6). The IC50 concentration was 45.69µg/ml for C and 86.16µg/ml for NC. Figure 7a and Figure 7b illustrate the direct microscopic views of drug-treated pictures for test chemicals C and NC, respectively. Statistical analysis for intragroup comparison with the untreated control indicated no significant changes at concentrations of 3.125ug and 6.25ug, but significant differences were observed at higher concentrations in the NC group. In the C group, an insignificant difference was observed solely at the 3.125 µg concentration, whereas significant differences were observed at higher concentrations, demonstrating increased cytotoxicity of C at elevated levels compared to NC. The intergroup comparison revealed statistically insignificant differences at lower concentrations of 3.125ug, 6.25ug, and 12.5ug; but exhibited a significant difference at higher concentrations of 25, 50, 100, 200, and 400ug.

Figure 6.

Percentage cell viability values against L929 cells after the incubation period of 24hrs of Nanocurcumin (NC) and Curcumin (C). X axis denotes compound concentration in µg/ml and Y axis denotes % cell viability. * Denotes p less than 0.05 versus the untreated control of the respective group

Figure 7.

Direct microscopic images of L929 cells treated with various concentrations of (a) Curcumin and (b) Nanocurcumin.

Discussion

Drugs delivered locally in periodontal pockets can augment results obtained by mechanical debridement and prevent recolonization of periodontal pathogens (21). Nanoparticle-based delivery is an effective method to enhance the bioavailability and solubility of C while protecting it from hydrolytic inactivation (22).

In the present study, the various characterizations of the prepared NC confirmed well-characterized nanoparticle formation. The enhanced water solubility of NC particles is attributable to increased surface area, which facilitates dissolution (23). Anbari et al. (24) indicated that decreasing the size of C particles enhanced solubility, bacterial absorption, and the capacity to inhibit methicillin-resistant S. aureus. UV-vis spectroscopy results indicated successful nanoparticle production, exhibiting an absorption peak at 430 nm, consistent with the findings of Kumar et al. (25). The increased intensity of the absorption peak of NC indicates its greater solubility relative to C.

Minor shifts in absorption peaks of the FTIR spectra of NC reveal the minimal structural difference between pure C and the synthesized NC, consistent with the study reported by Xie M et al.(26). SEM confirmed the presence of nanoparticles. Reduction in particle size enables easy diffusion into the gingival mucosa thus possessing increased antibacterial and anti-inflammatory potential compared to C. The absence of additional peaks in the 1H NMR spectrum reveals NC formation by C molecule aggregation with no impurities and the absence of chemical modification or degradation during NC preparation.

In the present study, NC exhibits lower MIC values than C, signifying superior antibacterial efficacy. Tetracycline served as the control in the MIC study because of its established antibacterial efficacy against several periodontal pathogens, including P. gingivalis and its therapeutic application in periodontitis (27). Maleki Dizaj S et al., (11) reported MIC and MBC values of C nanocrystals against clinically isolated P. gingivalis from patients with implant failure as 6.25µg/mL and 12.5µg/mL respectively. The values are consistent with the present study.

L929 mouse embryo fibroblasts were employed for cytotoxicity assessment in this investigation due to their well-characterized nature and ability to yield uniform and consistent results. This cell line is recommended for cytotoxicity assessment according to ISO 10993-5 guidelines (28) for the biocompatibility evaluation of dental materials and is suitable for preliminary biomaterial screening.

The cytotoxicity assessment on the L929 mouse embryo fibroblast cell line revealed that the proliferation and morphology of cells gradually decreased with exposure to higher concentrations of NC and C. This is in agreement with the study conducted by Hanna and Saad., (9), who reported that cell viability gradually decreased with increasing concentration of NC against normal baby hamster kidney cells and human epidermoid carcinoma-2 cells. The intergroup comparison revealed a statistically significant difference in cell viability from 25 µg/ml onwards, with NC demonstrating a significant reduction in cytotoxicity compared to C in higher concentrations. Thus, NC exhibits reduced cytotoxicity in comparison to C. There is a significant difference in IC50 values of NC and C, with NC demonstrating higher IC50 values (86.16 µg/ml) compared to C (45.69 µg/ml). According to ISO 10993-5 standards, a chemical is deemed cytotoxic if it decreases cell viability above 30% relative to untreated controls (28). Thus, concentrations above 12.5μg/mL of C and 50μg/mL of NC can be deemed cytotoxic from the present study.

The study's limitations include the exclusive collection of in vitro data, with cytotoxicity assessed in L929 fibroblasts, which may not accurately represent the effects of NC in clinical research, as mouse fibroblasts may demonstrate divergent cellular responses compared to human fibroblasts. The antibacterial activity results observed against the periodontal pathogen P. gingivalis cannot be generalized to a biofilm environment.

Conclusion

NC prepared in the present study exhibits water solubility, enhanced antibacterial efficacy, and reduced cytotoxicity relative to C. The cytotoxicity investigation demonstrated that NC is biologically safe at low concentrations. Analysis of antibacterial efficacy against P. gingivalis indicated that a reduction in the size of C particles lowered the MIC values, which is below the cytotoxic dose of NC. Additional research on the green synthesis of NC and its evaluation on experimental animals and further clinical trials should be undertaken to design and optimise appropriate scaffolds for controlled delivery of NC in the subgingival milieu.