ANTIMICROBIAL DRUG DISCOVERY OF A NOVEL QUATERNARY AMMONIUM SILANE: EFFECT ON BACTERIAL BIOFILMS, MACROPHAGES AND PERIODONTAL STEM CELLS

RANJEET AJIT BAPAT

Publication:
ANTIMICROBIAL DRUG DISCOVERY OF A NOVEL QUATERNARY AMMONIUM SILANE: EFFECT ON BACTERIAL BIOFILMS, MACROPHAGES AND PERIODONTAL STEM CELLS

dc.contributor.author	RANJEET AJIT BAPAT
dc.date.accessioned	2026-07-31T05:38:15Z
dc.date.available	2026-07-31T05:38:15Z
dc.date.issued	2025
dc.description.abstract	Background: Bacterial biofilms within the root canal system are a major contributor in the failure of endodontic treatment, primarily attributed to the presence of significant pathogens such as Enterococcus faecalis (E. faecalis), Fusobacterium nucleatum (F. nucleatum), and Porphyromonas gingivalis (P. gingivalis), which demonstrate considerable virulence, antimicrobial resistance, and the capacity to penetrate dentinal tubules. Conventional irrigants such sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) have limitations including cytotoxicity, inadequate biofilm eradication, and adverse responses including tissue irritation and precipitate formation. Quaternary ammonium silane (codename- K21), a functionalised organosilicon compound with antibacterial and anti-inflammatory properties, not only disrupts microbial biofilms but also preserves compatibility with host tissue components, including macrophages. Comprehensive research is warranted to confirm K21's therapeutic benefits since it has potential to block Sortase Av(SrtA) and matrix metalloproteinases (MMPs), enzymes engaged in bacterial adherence and tissue breakdown. Objectives: The aim of this research was to assess K21's antibacterial and anti-inflammatory performance as an endodontic irrigant. Among specific objectives were comparing 1.00% K21 with 0.5% K21, 2.00% CHX, 6.00% NaOCl, and saline on single-species (E. faecalis, F. nucleatum and P. gingivalis respectively) and dual-species (F. nucleatum and P. gingivalis) biofilms at 200 μm and 400 μm dentinal tubule depths. Additionally evaluated in the study were K21's effects on hPDLSCs survival as well as RAW 264.7 macrophage viability and anti-inflammatory (M2) polarization. Moreover, molecular simulations were performed to assess the interaction of K21 with SrtA and MMP-9 molecular structures. While the null hypothesis predicted no significant differences, the hypotheses postulated that 1.00% K21 would show better biofilm reduction, higher macrophage and hPDLSCs viability, and greater anti-inflammatory benefits than conventional irrigants. Methods: This multimodal investigation utilized a combination of in vitro assays and imaging techniques. Biofilms of E. faecalis, F. nucleatum, and P. gingivalis were cultured on dentin specimens and exposed to different irrigants: saline, 6.00% NaOCl, 2.00% CHX, 0.5%K21, and 1.00% K21. Colony-forming unit (CFU) counts were used to assess bacterial viability, while structural changes were analyzed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Raman spectroscopy was employed to identify chemical alterations in post-treatment biofilm components. Cell viability was evaluated using the MTT assay and mitochondrial staining in RAW264.7 macrophages, while the alamar blue assay was utilised for hPDLSCs. Additionally, the expression of cytokines (TNF-α, IL-1β, TGF-β, VEGF-A) associated with macrophage polarization was evaluated using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and gene expression analysis to determine the immunomodulatory impact of all the irrigants. Molecular simulations were carried out inside the Schrödinger Drug Discovery Suite using the Desmond package. Molecular simulations analyzed K21’s binding to MMP-9 and SrtA molecular structures with stability assessed via root mean square deviation (RMSD) and binding free energy (MM-GBSA). Statistical analysis used ANOVA with Tukey’s post hoc test (p < 0.05) and Pearson’s correlation for depth-dependent efficacy. Results: The CFU analysis revealed that K21, especially at a 1.00% concentration, consistently showed enhanced antibacterial efficacy relative to 6.00% NaOCl and was frequently comparable to or more effective than 2.00% CHX against E. faecalis, F. nucleatum, P. gingivalis, and dual-species biofilms. For CFU findings of E. faecalis, at a depth of 200 μm and a duration of 1-minute, 1.00% K21 exhibited the lowest CFU at 1.358 ± 0.193, in contrast to 0.5% K21 (3.453 ± 0.205), 2.00% CHX (4.65 ± 0.093), 6.00% NaOCl (5.64 ± 0.108), and saline (9.63 ± 0.031), with statistically significant differences (p < 0.001). At 400 μm and 1-minute, similar trends were observed. In 5-minute exposures, 1.00% K21 consistently exhibited the lowest CFUs (0.9687–0.9924), significantly surpassing 2.00% CHX and 6.00% NaOCl at both 200 μm and 400 μm (p < 0.001). For F. nucleatum biofilm, 1.00% K21 exhibited the lowest CFUs (2.1199 ± 0.20274 at 200 μm/5 min and 2.1782 ± 0.55736 at 400 μm/5 min), demonstrating significant reductions compared to 6.00% NaOCl (p < 0.001) and comparable efficacy to 2.00% CHX. At 1-minute application, CFU counts were markedly diminished by K21 group compared to 6.00% NaOCl. For P. gingivalis, at 200 μm/1 min, 1.00% K21 (3.9831 ± 0.10972) and 0.5%K21 were significantly better than 6.00% NaOCl (5.1798 ± 0.39327) and were comparable to 2.00% CHX (4.0792 ± 0.10074). At 400 μm/1 min and 5 min, all test irrigants demonstrated effectiveness relative to saline; nevertheless, 1.00% K21 consistently exhibited the lowest CFU counts with no statistically significant difference from 2.00% CHX. In dual-species biofilm, 1.00% K21 demonstrated significant reductions compared to saline at both 200 μm and 400 μm for 1 and 5-minute treatments. At 200 μm/5 min, 1.00% K21 (4.6173 ± 0.37708) and 0.5%K21 (4.7104 ± 0.34656) demonstrated superior efficacy compared to 6.00% NaOCl (5.1432 ± 0.48289). 1.00% K21 was comparable to 2.00% CHX (p > 0.05) and significantly better than saline (p < 0.001) and 6.00% NaOCl (p = 0.046). Morphological analyses utilising SEM and TEM corroborated the CFU findings, demonstrating that 1.00% K21 induced significant membrane rupture, cytoplasmic leakage, and biofilm matrix disintegration across all the three species. The 1.00% K21 treated groups exhibited the most pronounced intensity deterioration, and Raman spectroscopy examination indicated notable spectrum shifts in the 480–490 cm⁻¹ range, so corroborating the hypothesis of heightened structural disruption of bacterial components. For macrophage viability, all groups showed cell viability above 80%. While 0.5% and 1.00% K21 showed higher viability (87.17% and 87.4%), viability reduced for 6.00% NaOCl (82.42%) and 2.00% CHX (86.08%). K21 groups were noticeably better than 6.00% NaOCl, although all test groups had lowered viability relative to saline (p<0.05). Fluorescence imaging depicted strong, homogeneous mitochondrial signals maintained by saline and 0.5% K21 that indicated healthy, viable cells. The 1.00% K21 group maintained general function while only modestly altered mitochondrial structure. While 6.00% NaOCl produced severe cytotoxicity, low cell density, and weak fluorescence, 2.00% CHX exhibited variable effects with some mitochondrial fragmentation. At a concentration of 0.5%, K21 demonstrated superior mitochondrial preservation compared to 2.00% CHX and 6.00% NaOCl. SEM and TEM analysis indicated that macrophages treated with saline exhibited normal integrity and morphology. K21 exhibited electron-dense inclusions at elevated concentrations, signifying stress, but preserving cell morphology with slight surface alterations. The 6.00% NaOCl treatment caused severe structural damage, membrane fragmentation, and cytoplasmic debris; 2.00% CHX treated cells showed surface shrinkage and internal aggregates. K21 group displayed generally better cellular preservation than CHX and NaOCl groups. Gene expression analysis demonstrated that K21 induced modest upregulation of pro-inflammatory cytokines (TNF-α: ~4.24–4.26; IL-1β: ~1.43–1.89), whereas 2.00% CHX and 6.00% NaOCl strongly upregulated both TNF-α (~19.38 and 19.30) and IL-1β (~2.30 and 1.89). Conversely, K21-treated groups exhibited significantly higher anti-inflammatory markers, with VEGF-A reaching 58.93 ± 0.16 for 1.00% K21 and TGF-β at 6.51 ± 0.11, substantially exceeding levels observed with CHX (TGF-β: 6.31 ± 0.66) and NaOCl. Overall, K21 demonstrated a balanced immunomodulatory profile characterized by reduced inflammatory activation and enhanced pro-healing capacity compared to conventional irrigants. At 30 minutes of exposure, the viability of human periodontal ligament stem cells (hPDLSCs) remained approximately close to 80% for all tested irrigating agents. Specifically, 1.00% K21 demonstrated a viability of 81.13%, 0.5% K21 at 80.13%, CHX at 80.16%, and NaOCl at 79.34%. Saline showed the highest cell viability at 98.48%. High viability was indicated by SEM and TEM studies of hPDLSCs showing intact morphology and membrane integrity from saline-treated cells. Severe structural damage, membrane lysis, and cytoplasmic disintegration were produced by 6.00% NaOCl. Moderate membrane distortion and vacuolation caused by 2.00% CHX indicated cytotoxic effects. Although 1.00% K21 showed some structural changes while 0.5%K21 maintained better integrity. TEM verified that although K21 caused subcellular changes, it maintained membrane continuity, hence indicating better biocompatibility than 2.00% CHX and 6.00% NaOCl. The K21 group shows a specific pocket binding on several MMPs and specific SrtA structures, according to molecular simulation, which generates a classic clouting effect. This can prevent SrtA enzymes and MMPs from acting catalytically, avoiding structural changes in dentin. Conclusions The 1.00% K21 group demonstrated superior antibacterial efficacy against E. faecalis, F. nucleatum, and P. gingivalis in dentinal tubules at 200 μm and 400 μm depths compared to 6.00% NaOCl and saline, with performance comparable to 0.5% K21 and 2.00% CHX. In dual-species biofilms, 1.00% K21 outperformed NaOCl and saline while showing equivalent results to CHX and 0.5% K21. It exhibited significantly enhanced biocompatibility and anti-inflammatory properties in RAW 264.7 macrophages and hPDLSCs compared to NaOCl, with results comparable to 0.5% K21 and CHX. K21 promoted M2 macrophage polarization and a balanced immune response. Molecular docking revealed specific binding affinity to MMPs and Sortase A, indicating a unique "clouting effect" that enhances antibacterial and regenerative capabilities. Overall, 1.00% K21 represents a promising biocompatible, multifunctional endodontic irrigant with superior antibacterial and anti-inflammatory potential. Keywords: antibacterial, human periodontal ligament stem cells, K21, macrophages, molecular simulation, quaternary ammonium silane.
dc.identifier.uri	https://hdl.handle.net/20.500.14377/37846
dc.language.iso	en
dc.publisher	IMU University
dc.subject	Anti-Bacterial Agents
dc.subject	Biofilms
dc.subject	Macrophages
dc.subject	Stem Cells
dc.subject	Quaternary Ammonium Compounds
dc.title	ANTIMICROBIAL DRUG DISCOVERY OF A NOVEL QUATERNARY AMMONIUM SILANE: EFFECT ON BACTERIAL BIOFILMS, MACROPHAGES AND PERIODONTAL STEM CELLS
dc.type	Thesis
dspace.entity.type	Publication
oairecerif.author.affiliation	#PLACEHOLDER_PARENT_METADATA_VALUE#