Antimicrobial efficacy of triple antibiotic-loaded chitosan nanoparticles activated with photochemical disinfection: A microbiological and confocal microscopic analysis.

Introduction: This in-vitro study comprises Antimicrobial and Confocal Microscopic analysis of the efficacy of Triple Antibiotic and Toluidine Blue (TBO) loaded chitosan nanoparticles (chnp) activated with photodynamic therapy (PDT) against Enterococcus faecalis (Ef). Methodology: 105 single-rooted extracted teeth were decoronated, cleaned and shaped, and incubated with Ef. The roots were randomly divided into three groups: Group I-Control, Group II-Triple Antibiotic Paste (TAP), Group III: Triple antibiotic-loaded chitosan nanoparticles (tachnp), and Group IV: Tachnp and TBO loaded chnp activated with PDT. Ten specimens from all the groups were subjected to antimicrobial analysis and five specimens were observed under the confocal microscope after 24 h and 7 days. Group IV was additionally analyzed after 24 h and 7 days of laser activation.
Results: Group IV C showed the least colony-forming units followed by Group IVB, Group IIIB, Group IVA, and Group IIIA. Group II showed more colony-forming units. On intergroup comparison of mean colony count in control and experimental groups at 24 h and 7 days using one-way ANOVA F test was highly significant P < 0.001. The confocal microscopic images of IV C showed the increased intensity of fluorescent red indicating dead bacteria.
Conclusion: Within the limitations of this study, TAP loaded chitosan nanoparticles activated with PDT showed a significant reduction in colony count. Copyright:

INTRODUCTION

The ideal outcome of endodontic treatment is effective control of pathogenic microorganisms and mechanical stabilization of root canal dentin. Endodontics is an all-encompassing discipline that primarily seeks to understand the intricate root canal morphology and all of its complexities. The ability of bacteria to penetrate deep into dentinal tubules makes complete debridement almost impossible when the conventional method of endodontic instrumentation is performed to the highly skilled norms.[1]

Enterococcus faecalis (Ef), though makes a small proportion of the microbial flora in untreated canals, plays a vital role in the etiology of persistent infections and are resistant to traditional intracanal medicaments. The triple antibiotic paste (TAP), has gained a superior place in endodontics for the eradication of Ef.[23] Very recently, the triple antibiotic was utilized together with chitosan as a vehicle to increase its stability and controlled release of the medicament.[4] Chitosan is natural biopolymer that can be synthesized into nanoparticles (Np).[5] The hydroxyl and amino groups within the chitosan structure can be functionalized with the carboxyl group of proteins and photosensitizers.[6]

It is stated primafacie; photodynamic therapy (PDT) has been an adjunct to disinfection protocol due to increasing antimicrobial activity.[7] Reflecting on its evolution of development the focus is on the use of Np for photosensitizer delivery and release system. A combination of PDT and Chitosan nanoparticles (ChNp) was found to decrease microbial loading and protect dentin against collagenase.[8] Since various studies have been conducted with PLGA and ChNp as carriers of photosensitizer, Triple antibiotic-loaded chitosan nanoparticles (TAChNp) activated with PDT was evaluated as a novel approach with Diode Laser – 628 nm (Fotosan, CMS Dental, Copenhagen, Denmark) was considered.[91011]

MATERIALS AND METHODS

Preparation of Triple antibiotic paste loaded chitosan nanoparticles

Chitosan (4 g) were soaked overnight in 1% acetic acid (100 ml). Drug solutions of Ciprofloxacin, Metronidazole, and Minocycline were prepared separately as follows:

Ciprofloxacin (4 g) was dissolved in 50 ml of acetic acid

Metronidazole (4 g) was dissolved in 50 ml of 0.1N HCl

Minocycline (4 g) was dissolved in 50 ml of distilled water.

To the previously soaked chitosan solution, the drug solution as prepared above was added to get 1:1 ratio of chitosan: Drug and was stirred for 15 min. To this 1% tripolyphosphate (TPP) solution was added dropwise till the solution turned turbid and was stirred for 15 min at 500 rpm, then sonicated using probe sonicator for 30 min.

Preparation of toluidine blue ChNP

ChNp was prepared by drop wise addition of 15 ml TPP (1%) solution (4 g/100 ml). Toluidine Blue (TBO) (4 g) was dissolved in water (5 ml) to it and to it 500 mg ethylenediaminecarbodiimide and 950 mg N-hydroxylsuccinimide both sufficient to make 5 mM was added and stirred. This solution was added to the ChNp solution drop wise in a 1:1 ratio and was probe sonicated for 30 min and dialized against distilled water for 1 day by using cellulose membrane (Himedia, mol.wt 8000 Da).

The Np were freeze-dried at −45°C for 48 h using Labconco freeze dryer.

Characterization of chitosan nanoparticles

The particle size analysis was performed at 25°C using Melvin Zetasizer Nano ZS90 with laser diffraction with beam length 240 mm ranges, lens of 300RF mm, and 14.4% obscuration. The particle size of Np was between 313 nm to 385 nm. The Np solution was diluted in a 1:10 ratio with deionized water and zeta potential was analyzed using Universal dip cell. The particles carried zeta potential in the range of +20.8 mV and 29.8 mV and were able to entrap appreciable amounts (70%–78%) of the drug.

Preparation of tooth specimens

A total of 105 extracted single-rooted human teeth were collected for the study. The teeth were decoronated to standardize the length of the teeth to 14 mm and were instrumented with Protaper Universal (Dentsply, Mallieffer, Ballaigues) to an apical size of #40 (F4). The irrigation protocol was carried out with 5.25% NaOCl during the shaping of the canal. After shaping, the teeth were soaked in 17% ethylenediaminetetraacetic acid in an ultrasonic bath and activated for 2 min to remove the smear layer. The specimens were then autoclaved and subjected to bacterial contamination.

Culturing of Ef and Specimen contamination

Ef (ATCC 29212), from the slant provided by the National Chemical Laboratory, Pune, was grown on trypticase soy agar plate and observed under the microscope (BX-63 Olympus DIC) at 100% for purity of the culture. Single colonies were then inoculated in trypticase soy broth that was sufficient for the experimental teeth. Tooth specimens were placed individually in the tubes and incubated at 37°C for 4 weeks, maintaining the cell density at 2 × 108/ml.

Sample preparation

The teeth were washed with phosphate buffer saline (PBS) to remove the inoculation broth. The samples were then divided into three study groups and 1 control group.

Group I-Control group: No Triple antibiotic paste

GroupI I-Ciprofloxacin + Metronidazole + Minocycline + Macrogol +Propylene glycol

Group III-CiprofloxacinNp + MetronidazoleNp + MinocyclineNp + Macrogol + Propylene glycol

Group IV-CiprofloxacinNp + MetronidazoleNp + MinocyclineNp + TBO Np Macrogol + Propylene glycol.

Group I: Control; Group II: TAP (n = 30) were further divided into A (n = 15) and B (n = 15) which were evaluated after 24 h. and 7 days, respectively; Group III TAChNp (n = 30) – were further divided into two subgroups A (n = 15) and B (n = 15) which were evaluated after 24 h and 7 days, respectively. Among the 15 specimens, 10 were analyzed microbiologically and 5 were subjected to confocal microscopic observation.

Group IV: TAChNp and TBO-loaded Chitosan nanoparticles (TBOChNp) with PDT (n = 45) – were further divided into three subgroups A, B, and C with n = 15 in each group. In subgroup A, the specimens were activated using the Endo tip of the light-emitting diode laser at 628 nm for 30 s after 24 h. In Subgroup B, specimens were activated after 7 days. In Subgroup C, the specimens were activated after 24 h, the medicament was changed and again activated after 7 days.

For microbiological analysis, size #50 (F5) Protaper was introduced into the canal at 500 rpm and dentinal shavings were collected and placed in sterile Eppendorf tubes containing PBS. From each sample, 100 ml was serially diluted with PBS. Each dilution was spread on TSB plate and inoculated at 37°C for 24 h and the total viable count was calculated.

Five teeth from each group were randomly selected and prepared for observation under confocal Laser Scanning Microscope. Longitudinal grooves were placed with a diamond disc and split into two halves. The specimens were then placed in Eppendorf tubes with 1 ml PBS until stained for observation. Staining was carried out with L13152 Live/Dead BacLight Bacterial Viability Kit according to manufacturer's instructions and observed under confocal microscope within 15 min.

RESULTS

Table 1 depicts the results of the study, at 24 h. Group IVC showed the least colony-forming unit (210.8) followed by Group IIIA (261.1), Group IVA (279.3), and Group IIA (295.3). There was a significant difference between the groups P < 0.001** on one-way ANOVA “F” test.

Table 1

Intergroup comparison of mean colony count in control and experimental groups at 24 h

24 h (subgroup A)	Mean	SD	One-way ANOVA F-test value	P (significance)
Group IA (control)	314.7	7.45	100.02	<0.001**
Group IIA	295.3	12.91
Group IIIA	261.1	12.1
Group IVA	279.3	7.95
Group IVC	210.8	11.19

SD: Standard deviation, **P<0.001 -highly significant

Table 2 depicts pair-wise comparison between the groups using Tukey's post hoc test at 24 h. Group IVC (PDT at 24 h and 7 days) showed a highly significant difference.

Table 2

Pair-wise comparison of the groups using Tukeys post hoc test

Group	Comparison Group	Mean difference	P (significance)
Group IA (control) versus	Group IIA	19.4	0.006
	Group IIIA	53.6	<0.001**
	Group IVA	35.4	<0.001**
	Group IVC	103.9	<0.001**
Group IIA versus	Group IIIA	34.2	<0.001**
	Group IVA	16.0	0.037
	Group IVC	84.5	<0.001**
Group IIIA versus	Group IVA	18.2	0.012*
	Group IVC	50.3	<0.001**
	Group IVC	68.5	<0.001**
Group IVB versus	Group IVC	37.1	<0.001**

*P<0.05 - significant, **P<0.001 - highly significant, P>0.05 - not significant

Table 3 depicts the results of the study at 7 days. Group IVC showed the least colony-forming units (198.3) followed by Group IVB (212.2), Group IIIB (245.3), and Group IIB (278.1). There was a significant reduction in group IVC (PDT at 24 h and 7 days) and group IVB (PDT at 7 days), the intergroup comparison was significant P < 0.001*.

Table 3

Intergroup comparison of mean colony count in control and experimental groups at 7 days

7 days (subgroup B)	Mean	SD	One-way ANOVA F-test value	P (significance)
Group IB (control)	293.8	7.16	257.067	P<0.001**
Group IIB	278.1	10.65
Group IIIB	245.3	6.18
Group IVB	212.2	6.12
Group IVC	198.3	5.07

SD: Standard deviation, **P<0.001 -highly significant

Table 4 depicts pair-wise comparison between the groups using Tukey's post hoc test at 7 days. Group IVC (PDT at 24 h and 7 days) showed highly significant difference. showed highly significant difference as P<0.001**.

Table 4

Pair-wise comparison using Tukey’s post hoc test

Group	Comparison Group	Mean difference	P (significance)
Group IB (control) versus	Group IIB	15.7	<0.001**
	Group IIIC	48.5	<0.001**
	Group IVB	81.6	<0.001**
	Group IVC	95.5	<0.001**
Group IIB versus	Group IIIB	32.8	<0.001**
	Group IVB	65.9	<0.001**
	Group IVC	79.8	<0.001**
	Group IVB	33.1	<0.001**
	Group IVC	47.0	<0.001**
Group IVB versus	Group IVC	13.9	<0.001**

* P<0.05 – significant, **P<0.001 - highly significant, P>0.05 - not significant

Table 5 depicts Intra-group comparison of mean colony count in control and experimental groups between 24 h. (Subgroup A) and 7 days (Subgroup B) using unpaired t-test the difference was highly significant for Group I, IVA, and IV B. P < 0.001** and was significant for group IVC P < 0.005*.

Table 5

Intragroup comparison of mean colony count in control and experimental groups between 24 hours observation (subgroup A) and 7 days (subgroup B) observation period

	24 h (subgroup A)		7 days (subgroup B)		Unpaired t-test value	P (significance)
	Mean	SD	Mean	SD
Group IA (control)	314.7	7.45	293.8	7.16	6.394	<0.001**
Group IIA	295.3	12.91	278.1	10.65	3.249	0.004
Group IIIA	261.1	12.1	245.3	6.18	3.676	0.002
Group IVA	279.3	7.95	260.5	7.53	5.426	<0.001**
Group IVB	247.9	15.98	212.2	6.12	6.596	<0.001**
Group IVC	210.8	11.19	198.3	5.07	3.216	0.005*

*P<0.05 - significant, **P<0.001 - highly significant, P>0.05 - not significant. SD: Standard deviation

Table 6 depicts presence and absence of discoloration in the groups – Group IVB and group IVC showed discoloration this is due to be TBO, however group IVA which also contained TBO dye did not show discoloration at 24 h.

Table 6

Discoloration in the groups

Groups	Discoloration
Group IA	Absent
Group IB	Absent
Group IIA	Absent
Group IIB	Absent
Group IIIA	Absent
Group IIIB	Absent
Group IVA	Absent
Group IVB	Present
Group IVC	Present

Confocal Microscopic observation reveals the increased intensity of fluorescent green in Group 1A, 1B, 2A, 3A after 24 h indicating the presence of live bacteria [Figure 1a, 2a, 3a]. The presence of red fluorescent zone indicating dead bacteria was seen after 7 days in all the three groups [Figures 2b, 3b, 4b and c]. In GroupIII (TAChNp), this region was enhanced penetrating deep into the dentinal tubules [Figure 3b]. The intensity of this zone was more pronounced with GroupIV (PDT) indicating better efficiency after 24 h and 7 days of laser activation [Figure 4a-c].

Figure 1

Group I (Control): No Triple Antibiotic Paste (a: 24 h, b: 7 days) green florescence indicating live bacteria

Figure 2

Group II-Triple antibiotic paste (a: 24 h, b: 7 days) green florescence indicating live bacterial and red florescence indicating dead bacteria

Figure 3

Group III-Triple antibiotic loaded chitosan nanoparticles (a: 24 h, b: 7 days) green florescence indicating live bacterial and red florescence indicating dead bacteria

Figure 4

Group IV-tachnp and toluidine blue loaded chnp activated with photodynamic therapy. (a: 24 h, b: 7 days, c: 24 h and 7 days activated with changed dressing) green florescence indicating live bacterial and red florescence indicating dead bacteria

Statistical analysis

The intergroup comparison was made using one–way ANOVA “F” test. The pairwise comparison was made using Tukey's post hoc test. The intragroup comparison was done using unpaired “t” test.

DISCUSSION

Effective root canal disinfection is undoubtedly a fundamental component of successful root canal treatment. With the insight to combat with the challenge of delivering the medicament to inaccessible areas, triple antibiotics and photosensitizer were loaded into the chitosan Np in this study. Chitosan, the natural polymer obtained by deacetylation of chitin, gained more attention as drug delivery carriers because of better stability, low toxicity, simplest mild preparation, and versatile routes of administration.[1213] The Np were prepared by the Ionotropic gelation method, based on chitosan microparticle technology.[14]

TAP along with chitosan showed increased antimicrobial efficacy against Ef and Candida albicans.[4] In an animal experimental study by Hoshino et al.[15] the intracanal drug delivery solution of TAP resulted in >99% of reduction in the colony-forming unit levels.[16] In an study conducted by Shaik et al. combining chitosan Np with TAP or calcium hydroxide effectively reduced the Ef count.[4] The findings of our study are consistent to the previously reported, where the effectiveness of intracanal medicament is enhanced by the addition of chitosan Np.[416]

The recent focus of PDT to use Np photosensitizer delivery and release system has shown promising results in the reduction of bacterial count having an added advantage of no thermal effects, enhanced crosslinking of collagen fibrils in dentin matrix, and inhibit collagenolytic activity.[17]

The results of this study showed increased bacterial reduction and statiscally significant difference in GroupIII (TAChNp) compared to control. The confocal imaging which also shows red fluorescence extending deep in the dentinal tubules signifying increased depth of penetration of the medicament when used as Np. The laser activated group also showed significant reduction (P < 0.001) and increased intensity of red fluorescent zone compared to the control group. This was more enhanced in Subgroup C where laser activation was done after 24 h and 7 days. The specimens in Group IV, where the medicament was left for 7 days absorbed the stain of TBO dye from the TBOChNp, suggesting that the TBOChNp should not be left in the canal for more than 24 h. An alternative method of placing the TBOChNp just before the laser activation or using a different photosensitizer can be considered. Ng et al. in their ex vivo study compared microbiological samples from necrotic root canals of recently extracted human teeth before and after treatment with CMD alone or in combination with antimicrobial PDT and reported significant microbial reduction after antimicrobial PDT and less microbial concentrations in dentinal tubules at a depth up to 485 μm.[1] In a study by Mustafa et al. PDT was more effective against Ef and C. albicans.[18] In a review by Abdelkarim-Elafifi et al. PDT showed promising results against biofilms disruption.[19] In a randomized controlled clinical trial by Asnaashari et al. the effectiveness of a single-visit approach using PDT showed superior microbiological eradication when compared with calcium hydroxide dressing in two sessions.[20] There are less studies reporting the antimicrobial effectiveness of triple antibiotic Np activated with PDT, this study is distinctive and reports superior antimicrobial efficacy of triple antibiotic Np activated with photochemical disinfection using both microbiological and confocal testing.

Newer studies should be carried out with different mode of activation of TAChNp since this proved to be a good alternative to the TAP in terms of bacterial reduction and increased depth of penetration moreover it is safe and predictable. More studies and clinical trials are needed with long-term follow-up to authenticate the effectiveness of PDT as an adjunctive tool.

CONCLUSION

Within the limitations of this study, it can be concluded that TAChNp showed better results compared to TAP (Control). TAChNp and TBOChNp activated with PDT caused a definite reduction in colony count. Clinical studies should be conducted to correlate the results of this in-vitro study.

Statement of clinical relevance

This article emphasis on an innovative approach of Triple antibiotic nanoparticles activated with photo dynamic therapy aiding in better elimination of one of the most resistant bacteria in the root canal system that is E. faecalis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.