Clinical Oral Investigations Biological and Physicochemical Characterization of Nanoemulsions Containing Morin and its Derivatives for Endodontic Purposes

Clinical Oral Investigations Biological and Physicochemical Characterization of Nanoemulsions Containing Morin and its Derivatives for Endodontic Purposes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical Oral Investigations Biological and Physicochemical Characterization of Nanoemulsions Containing Morin and its Derivatives for Endodontic Purposes Jesse Augusto Pereira, Rafael Araújo Rios, Jonatas Lobato Duarte, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5923566/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives Considering the lack of medications with a wide range of therapeutic effects for the endodontic treatment of immature teeth, this study aimed at synthesizing two morin (Mo) derivatives and testing their cytotoxicity and effect on multispecies biofilm in solution and loaded in nanoemulsions (NE). Material and methods Minimum inhibitory and bactericidal concentration (MIC/MBC) of Mo, penta-acetylated Mo (Ac-Mo), Mo complexed with strontium (Sr-Mo) and control chlorhexidine (CHX) were determined against some oral bacteria. NE were physiochemically characterized by analysis of particle size, polydispersity index and zeta potential. NE containing Mo, its derivatives and CHX were evaluated against multispecies biofilms by bacterial counts, scanning electron microscopy and confocal microscopy. The cytotoxicity of the compounds and NE was also determined in fibroblasts using resazurin assays. The data were statistically evaluated (p < 0.05). Results All compounds presented bactericidal action against all bacteria tested (MIC/MBC from 0.125 to 1 mg/mL). Metabolic activity of fibroblasts was higher than 70% after treatment with compounds at 0.25mg/mL or lower. NE showed good droplet size distribution in nanoscale and stability. The highest bacterial reduction in multispecies biofilms was observed in NE + Ac-Mo, followed by NE + Mo, CHX and NE + Sr-Mo groups. All NE diluted at 12.5% did not affect fibroblast metabolism after 24h of treatment. Conclusions Morin and its derivatives, either alone or loaded in nanoemulsions, were effective against oral bacteria and showed cytocompatibility at lower concentrations. Clinical Relevance: Nanoemulsion containing penta-acetylated morin could be an alternative intracanal medication for reducing residual bacteria between clinical appointments in regenerative endodontic approaches for immature teeth. antimicrobial activity oral biofilms cytotoxicity morin nanoemulsions Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Treating young permanent teeth with irreversible pulp damage is a significant challenge for endodontists and pediatric dentists. Since the root canals and apices of the permanent teeth are still not completed, these processes can be interrupted due to trauma or infection [ 1 , 2 ]. The conventional treatment, or apexification, involves the removal of vital or necrotic pulp, disinfection, and induction of a calcified barrier in the roots by using calcium hydroxide as an intracanal medication. However, this procedure does not increase the root length and strength, leading to a higher risk of reinfection and tooth fracture [ 3 ]. New approaches of regenerative endodontics for immature teeth have included minimal or no instrumentation of the dentinal walls and disinfection with effective but non-toxic irrigates/intracanal medicaments to solve the signs and symptoms of pulpal or periapical diseases and allow tissue regeneration [ 1 , 2 , 4 – 6 ]. The irrigation solution most used for root canal disinfection is sodium hypochlorite (NaOCl), with concentrations varying from 0.5 to 6%, for its high antimicrobial efficacy and tissue dissolution capacity [ 7 – 9 ]. NaOCl in concentrations from 1 to 5.25% were similarly effective in reducing Enterococcus faecalis in root canals since large quantities and regular irrigant exchanges are maintained, especially for lower concentrations [ 8 ]. Considering the cytotoxicity, NaOCl from 0.5 to 3% reduced 37% of stem cells of the apical papilla (SCAP) survival in an organotypic root canal model; however, after final irrigation with 17% EDTA and saline solution, cell viability was recovered [ 9 ]. Another important antimicrobial agent in endodontics is chlorhexidine, a bisbiguanide with a wide-range effect against Gram-positive and Gram-negative bacteria, fungi, and viruses [ 10 , 11 ]. Some studies have demonstrated a higher effect of chlorhexidine in eliminating E. faecalis from dentin tubules than other medicaments [ 10 – 12 ]. Conversely, chlorhexidine has presented cytotoxicity when in contact with odontoblastic cells, even at low concentrations [ 13 ]. Manual syringe irrigation is still the most popular method; however, it does not remove debris from secondary canals and apical irregularities. The use of auxiliary means to increase irrigation efficacy, such as passive ultrasonic activation (PUI), can increase the removal of debris, organic tissues, and microorganisms from the root canals. PUI consists of transmitting acoustic energy from an oscillating file or smooth wire to an irrigant solution in the root canal, facilitating the penetration into unassessed root canal areas [ 14 , 15 ]. Previous studies have shown that PUI was more effective in removing dentin and pulp remnants and reducing planktonic bacteria content than conventional irrigation [ 14 – 16 ]. A natural polyphenol called morin (3, 5, 7, 20, 40-pentahydroxyflavone) is found in plants such as Prunus dulcis (almonds), Maclura tinctoria (figs) and other plants in the Morceau family, such as Psidium guajava (Indian guava) [ 17 ]. Morin has demonstrated an inhibitory effect against several oral pathogenic microorganisms [ 18 ] and reduces the ability of adhesion and biofilm formation by Streptococcus mutans [ 19 ]. Some studies have synthesized morin derivatives, searching for new compounds with better antimicrobial activity than morin. Sodium salt of morin-5'-sulfonic acid showed an effect against antibiotic-resistant bacteria and clinical isolates of S. aureus with lower minimal inhibitory concentration (MIC) values than morin but higher MIC values against E. faecalis and Pseudomonas aeruginosa strains [ 20 ]. Morin has also been complexed with ions, such as lanthanum (III), gadolinium (III) and lutetium (III) and demonstrated inhibitory action against E. coli , Klebsiella pneumoniae , S. aureus at different concentrations [ 21 ]. Morin complexed with strontium (Sr + 2) reduced the differentiation of pre-osteoclasts and maintained the mineralization ability of osteoblasts compared to noncomplexed morin and strontium ranelate at the same concentrations [ 22 ]. The administration of phytomedicines generally presents limitations relative to their low aqueous solubility, low permeability, high molecular weight, enzymatic degradation, low bioavailability, and drug stability [ 23 ]. Nanoemulsions are considered a high-standard topical drug delivery by controlling drug release and focusing on site-specific action [ 24 ]. Nanoemulsions are oil-in-water (o/w) or water-in-oil (w/o) dispersions of two immiscible liquids that have been stabilized using a suitable surfactant. The following characteristics are mandatory to develop nanoemulsions: droplet size of 1-100 nm; mixing two fluids with surfactant; protection of medicines from physical and chemical degradation; being non-toxic; increasing the solubility of medicines; and facilitating the distribution of many medicines [ 24 , 25 ]. Cinnamic acid-loaded nanoemulsion was compared to pure cinnamic acid. It showed a higher effect in inhibiting planktonic cells and biofilms of S. typhimurium , S. aureus and P. aeruginosa due to the subcellular size of nanoemulsion that allowed it to reach bacterial targets effectively [ 26 ]. Some studies have demonstrated that morin in muco-adhesive controlled-release systems (gellan gun-based) effectively controlled single-species and multispecies biofilms [ 27 , 28 ]. Given the limited research on new compounds that have a wide range of therapeutic effects and drug delivery systems for endodontic applications, especially for treating immature teeth, this study aimed to achieve two main objectives. First, the study sought to synthesize two morin derivatives and test their antimicrobial and cytotoxic effects. Second, the study aimed to develop and characterize nanoemulsions containing morin and two morin derivatives, and then evaluate their cytotoxicity and effect on multispecies biofilms. The null hypothesis was that morin derivatives alone or loaded in nanoemulsions did not exhibit antimicrobial or antibiofilm activity and cytocompatibility. Material and Methods Unless otherwise specified, compounds and reagents were purchased from Sigma-Aldrich® (St. Louis, MO, USA), bacterial culture media from Difco (Kansas City, MO, USA) and culture media/supplements for cell cultures from ThermoFisher Scientific® (Waltham, MA, USA). Synthesis of penta-acetylated morin (Ac-Mo) An organic synthesis reaction for morin acetylation was carried out as previously described in Guimarães et al. [29] with some modifications. Briefly, morin hydrate (500mg, Sigma #M4008) was dissolved in 20mL acetic anhydride and 20mL pyridine in a round bottom flask and was heated between 94°C and 100°C in an oil bath. The reaction was maintained for 48h and monitored by analytical thin-layer chromatography (TLC) plates, using ultraviolet light (at 254 and 365 nm) and in the chemical agent sulfuric anisaldehyde. After the conversion of morin, the mixture was poured onto a beaker with crushed ice and stored in a refrigerator for 24 hours. The precipitate was vacuum filtered and left to dry at room temperature, presenting as a brown powder. For purification, the crude product (0.628mg) was subjected to a chromatography column over silica gel, using a mixture of the solvent's hexane and ethyl acetate in a 60:40 ratio, yielded around 56 fractions. These fractions were subjected to analytical thin-layer chromatography for a first purification analysis. The purification process by column chromatography was repeated, yielding around 145 fractions subjected to analytical thin-layer chromatography. 1H-NMR and 13C-NMR spectra were obtained on a Bruker Avance III HD (Bruker, Germany) at 400 MHz. Synthesis of strontium-morin complex (Sr-Mo) The synthesis of Sr-Mo was performed according to Cruz et al. [22]. Morin hydrate (0.150g) was solubilized in 50.0 mL methanol with 100 mL of NH4OH in a round bottom flask under continuous stirring. After that, SrCl2.6H2O (0.399g) was slowly added, and the mixture was kept under agitation for 2 h. Then, the product was centrifuged, washed with chloroform: t-butanol solution (1: 1, v/v) and dried at 37oC. The complete structural characterization of the final product is described elsewhere [22]. Stock solutions and controls Stock solutions of morin and their derivatives were prepared and frozen in dimethyl sulfoxide – DMSO at 30mg/mL. Only work solutions (in culture medium) containing up to 1% DMSO were used for the subsequent tests so as not to influence the results. For microbiological and cytotoxicity assays, chlorhexidine digluconate (Farmácia Manipullis, Araçatuba – SP/BR) in deionized water was chosen as a positive control (antimicrobial agent considered as a gold standard in Dentistry), and nanoemulsion with no compounds (NE) and culture media (Control) were determined as controls. All compounds were sterilized by filtration through a 0.22 µm membrane filter. Suppl. Table 1 presents the chemical structure, empirical formula, and molecular weight of morin and its derivatives. Unless otherwise mentioned, the assays were performed in triplicate in three independent days (n = 9). Determination of antimicrobial activity and cytotoxicity of compounds Standard strains and growth conditions The following bacterial strains were donated by Oswaldo Cruz Foundation (FIOCRUZ, RJ, Brazil) for this study: Enterococcus faecalis (ATCC 51299), Actinomyces israelii (ATCC 12102), Streptococcus mutans (ATCC 25175), Lactobacillus casei (ATCC 393), and Fusobacterium nucleatum (ATCC 25586). The culture media used for the growth of each bacterial species were as follows: Mitis Agar Salivarius Agar with 0.2 U/mL bacitracin for S. mutans , MRS Rogosa Agar for L. casei , Brain Heart Infusion Agar – BHIA for A. israelii and E. faecalis. F. nucleatum was grown in BHIA with 5mg/L of hemin, 5mg/mL of menadione and 5% of defibrinated sheep blood. All plates were incubated at 37°C under anaerobic conditions (AnaeroGen; Oxoid, Hampshire, UK). Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The MIC assays were measured using the microdilution method, based on the CLSI standards [30], with some modifications [31]. Briefly, cultures of the bacterial species mentioned previously were subcultured in Miller Hinton (MH) broth medium for 24h at 37° C. Then cells were adjusted to obtain 1-5x 105 cells/mL in MH. Counts were validated by counting on MH agar. The inoculum of each bacterium was then incubated in a serial dilution (from 1 to 0.0001 mg/mL) of each compound - Mo, Ac-Mo and Sr-Mo and controls dissolved in sterilized water. After incubation for 24 h (and 48h for F. nucleatum ) at 37°C, the microtiter plates were stained with resazurin solution at 5 µg/mL for 3h and analyzed at 570 and 600 nm in a spectrophotometer (Biotek, Winooski, VT). Bacterial suspensions were incubated with chlorhexidine (CHX) or without any antimicrobial agent as positive and negative controls, respectively. The MIC was defined as the lowest concentration of the compound in which there was no detectable growth. Media containing the MIC (and two higher concentrations) were serially diluted, plated on MH agar, and incubated at 37°C for 48h. The number of colony-forming units/ml (CFU/mL) was determined by a binocular stereomicroscope. MBC was considered when the compound killed more than 99.9% of the bacteria. Cytotoxicity of the compounds Metabolic activity was determined on culture of murine fibroblasts L929 fibroblasts, according to Duque et al. [32]. Cells were grown in Dulbecco's modified Eagle medium (DMEM, Dulbecco's Modified Eagle's) supplemented with 10% fetal bovine serum and 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine containing 5% CO2 and 95% air at 37°C until reaching 80% confluence. Then, cells were cultured at a density of 5x 104 cells/well in 96-well plates and incubated for 24 h. The cells were treated with serial dilutions (from 1 to 0.0019 mg/mL) of Mo, Ac-Mo, Sr-Mo and CHX for 24 h. Cells were washed with PBS, and resazurin 70µM in DMEM was added for 4h. After that, the plates were analyzed in a spectrophotometer at 570 and 600nm. The final values were obtained by the subtractions between the absorbance values at both wavelengths. They were converted into a percentage of cell viability, considering the growth in the DMEM medium to be 100% and the means determined for each group. Preparation and characterization of nanoemulsions The preparation of nanoemulsions (NE) was performed according to Bouchemal et al.; 2004 with some modifications [33]. The oil phase composed of medium-chain triglycerides (5% w/w) was weighed together with each compound (Mo, Ac-Mo and Sr-Mo) at 2 mg/mL and polysorbate 20 (5% w/w) and kept under agitation in a magnetic stirrer overnight. The aqueous phase was composed of water q.s.p. (10 g), which was added dropwise onto the oil phase, thus forming a pre-emulsion. This mixture was sonicated for 5 minutes, with a pulse of 1 min, pulse of 30, and amplitude of 27. The formulations were kept at room temperature and evaluated visually after 24h. Then, they were diluted in deionized water (1:25), and NE's average hydrodynamic diameter and polydispersity index (PDI) were determined using dynamic light scattering. Zeta potential was determined by electrophoretic mobility in a Zetasizer 3000 HSA (Malvern Instruments) device, using a 10mW HeNe laser at 633nm (173o of detection angle) at 25 o C in automatic mode. NEs were kept in the refrigerator at 4 C until used. Biological characterization of nanoemulsions Effect of NEs on multispecies biofilms and scanning electron microscopy The multispecies biofilm assays were conducted by mixing E. faecalis , A. israelii , L. casei , S. mutans and F. nucleatum in equal aliquots in BHI broth containing 1% glucose [31]. Briefly, sterile 96-well U-bottom polystyrene microplates were pre-treated with 150 µL of artificial saliva (800mL of deionized water, 1.6 g of yeast extract, 4 g of peptone, 0.28 g of NaCl, 4 g of sucrose, 0.16 g of CaCl2, 0.16 g of KCl and 0.8 g of mucin) for a period of 4 h at 37°C in the 5% CO₂ (coating phase) [34]. Bacterial cultures were grown individually for 24h; then, they were centrifuged and washed in PB buffer, and the cell number was adjusted to 1-5x103 CFU/mL in BHI broth containing 1% glucose. Then, they were mixed in equal aliquots and inoculated in the microplates. After 7 days of growth in anaerobic conditions, biofilms were washed twice with sterile saline solution, and 100 µl of NE, NE + Mo, NE + Ac-Mo, NE + Sr-Mo and CHX at 0.5 mg/mL (100x the highest MIC) was inserted into each well (n = 6). The plates were incubated for 24h at 37°C in anaerobic conditions. After scraping of biofilms, aliquots from all wells were resuspended, serially diluted, and plated on BHI agar. The plates were incubated for 48h for further counting of CFU/mL [31]. The same experiments were conducted in parallel in coverslips for scanning electron microscopic (SEM) analysis. The samples were dehydrated by washing in a series of ethanol (70% for 10min, 95% for 10 minutes, and 100% for 20 minutes) and air-dried in a desiccator. Afterwards, coverslips were mounted into aluminum stubs, sputter coated with gold, and analyzed in a scanning electron microscope (Leo, Cambridge, MA, USA) [35]. Effect of NEs on the multispecies biofilms formed on human root dentin and analysis by Confocal Microscopy Specimens' preparation and dentin contamination This methodology was carried out according to Andrade et al. [36] with some modifications. Human lower permanent incisors were stored in saline solution after cleaning and disinfection in 1% sodium hypochlorite solution for 48h (Ethical committee approval: CAAE − 38042320.6.0000.5420). The crowns and apices were removed, and the length of the roots was standardized at 12mm from apex to coronal edge using a 0.6 mm diamond disk (Isomet 5000; Buehler Ltd, LakeBluff, IL) at 1000 rpm, under irrigation with water. Root canals were assessed with a #6 spherical bur and instrumented with reciprocating files (Reciproc R25; VDW, Munich, Germany), considering the total canal length under irrigation with saline solution. Afterwards, specimens were immersed in tubes with 17% EDTA for 3 min, followed by 5 min in deionized water in an ultrasonic bath. The specimens were sterilized in an autoclave at 121°C for 15 min. Then, they were inserted into a tube containing 1mL BHI (w/hemin and w/menadione) with the canal side up and kept in an ultrasonic bath for 3 min to allow the culture medium to enter the dentinal tubules. Dentin specimens were contaminated by mixing E. faecalis , A. israelii , L. casei , S. mutans and F. nucleatum in equal aliquots in BHI broth containing 1% glucose, as mentioned before. The tubes were centrifuged at 1400xg, 2000 xg, 3600 xg and 5600 xg in this sequence, twice each, for 5 minutes. A fresh aliquot of bacterial cultures was added between each centrifugation, and the old one was discarded. All microtubes were incubated at 37°C in BHI broth for 14 days under anaerobic conditions to form multispecies biofilms inside dentin tubules. The medium was changed every 72 hours. The dentin specimens were removed from the microtube and washed with sterile water for 1 min. The external surfaces (cementum side) of the specimens were closed with nail polish. Protocols of canal treatment and confocal analysis All dentin specimens were fixed with double-sided tape in 24 well microplates, and their canals were irrigated with 1mL of 0.5% sodium hypochlorite for 1min (twice), followed by aspiration and new irrigation with 1mL of 17% EDTA for 2 min and 1mL of saline solution for 2 min. Then, the specimens were randomized and divided into groups: control – saline solution; NE (no antimicrobial agents), NE + Mo, NE + Ac-Mo, NE + Sr-Mo (at 2mg/mL) and CHX at 0.5mg/mL. Root canals were filled with 1mL NE and activated with ultrasonic smooth wire (Irri sonic; Helse Dental Technology, Brazil) placed 1.0 mm short of the working length and activated using 3 cycles of 20 seconds) for 1 min. Specimens were then incubated at 37°C for 24h in sealed microplates to avoid dryness. After that, root canals were dried with paper points and immersed in a solution of 0.9 mM KH2PO4 and 1.5 mM CaCl 2 (pH 7.0) for 1 minute to avoid residual effects of the treatments. Then, the specimens were longitudinally sectioned and stained with the fluorescent LIVE/DEAD Backlight Bacterial Viability stain (Molecular Probes, Eugene, OR). The fluorescence of the stained cells was evaluated in a Leica TCS SPE confocal microscope (Leica Microsystems, Germany), and the 2D images (3 specimens and n = 6 images/per group) acquired by the confocal software using the resolution of 1024x1024 pixels. Emission dead (avital) cells stained in red were visualized at 620nm, and emission vital cells stained in green were visualized at 530nm [37]. The proportion of emission dead cells was determined by counting emission dead cells over total cell counts (vital + dead cells) using the Image J 1.48 program (NIH, Bethesda, MA, USA) [31]. Cytotoxicity of NE The cytotoxicity of NEs was determined on the culture of murine fibroblasts L929 fibroblasts, following the methodology previously described (item Cytotoxicity of the compounds) . NE were diluted 10x before being applied on cell cultures. The cells were treated with serial dilutions of NE, NE + Mo, NE + Ac-Mo and NE + Sr-Mo (from 50 to 6.25%) for 24 h. Cells were washed with PBS, and resazurin 70µM in DMEM was added for 4h. After that, the plates were analyzed in a spectrophotometer at 570 and 600nm. The final values were obtained by subtracting the absorbance values at both wavelengths, and they were converted into the percentage of cell viability, considering the growth in the DMEM medium to be 100% and the means determined for each group. Statistical analysis Statistical analysis was accomplished using JAMOVI software (version 2.5.7). Datasets were assessed for analysis of variance (one-way or two-way ANOVA), followed by Tukey's post hoc test. The significance level was set at p < 0.05. Data were displayed as mean ± standard deviation (SD). Results Table 1 shows the values of minimal inhibitory and bactericidal concentrations (MIC/MBC) in mg/mL determined for morin (Mo) and their derivatives (Ac-Mo and Sr-Mo) against some oral bacteria. All compounds were bactericidal, presenting values of MIC from 0.125 to 0.5 mg/mL and MBC from 0.25 to 1 mg/mL. Ac-Mo presented values of MIC lower than Mo for E. faecalis, A. israelii and S. mutans . The values of MIC/MBC for Sr-Mo were higher than Mo when tested on S. mutans and F. nucleatum . CHX had the lowest MIC/MBC values for all bacteria tested. Table 1. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) in mg/mL for the flavonoids and control chlorhexidine against the oral microorganisms tested. E. faecalis A. israelii S. mutans L. Casei F. nucleatum Morin (Mo) 0.5 (1)* 0.25 (0.5) 0.25 (0.5) 0.125 (0.5) 0.125 (0.25) Acetylated Morin (Ac-Mo) 0.25 (0.5) 0.125 (0.25) 0.125 (0.25) 0.125(0.25) 0.125 (0.25) Sr–morin complex (Sr-Mo) 0.5 (1) 0.25 (0.5) 0.5 (1) 0.25 (1) 0.5 (1) Chlorhexidine 0.004 (0.009) 0.0001 (0.004) 0.0001 (0.001) 0.0001 (0.001) 0.0003 (0.0006) *In parentheses - MBC: >99.9% cell reduction. MIC results were based on resazurin staining and MBC results were based on CFU/mL count in Miller Hinton Agar (MHA) medium. The growth of microorganisms without antimicrobials in MHA was considered 100%. In Suppl. Figure 1A signs 2.34; 2.33; 2.32; 2.19 and 2.17 represent the five resonance signals of the five methyls, thus confirming that morin was penta-acetylated. Likewise, in Suppl. Figure 1 B , resonance signals 21.38; 21.32; 21.23; 21.05 and 20.42 represent the five methyls, thus confirming that the penta-acetylation was successful and confirmed through the two experiments. Using NMR spectra, it was determined that the acetylated morin had a good purity level and could proceed to the testing phase. Figure 1 presents the metabolic activity (%) of fibroblasts (NHI/3T3) after 24h treatment with different concentrations of Mo, Ac-Mo, and Sr-Mo. At 1mg and 0.5 mg/mL, metabolic activity varied from 21.94 to 56.07% and from 50.75 to 62.52%, respectively, being statistically higher for Mo derivatives. All groups differed from the control group (DMEM). From 0.25 mg/mL, all compounds were considered as cytocompatible since metabolic activity was higher than 70% (Mo: 79.14%; Ac-Mo: 119.34% and Sr-Mo: 94.59%). At this concentration only Mo and Ac-Mo statistically differed from the control. CHX was cytotoxic at all concentrations tested. NE+Mo, NE+Sr-and NE+Ac-Mo showed a yellowish, brownish, and white color appearance, similar to compounds free, respectively, as observed in Suppl. Figure 2 . Considering the characterization of nanoemulsions shown in Table 2 , the results for particle size were similar among the groups. PDI is an index that assesses the relative homogeneity in the sizes of particles distributed in samples, and it was calculated by dividing the average droplet size by an average number of measured droplets. Light-scattering analysis showed mean PDI values of 0.086 for NE containing Mo and Sr-Mo after formulation and monomodal distribution. For NE+Ac-Mo and NE-Sr-Mo, PDI was lower than the other NE. This parameter directly reflects droplet size homogeneity in the NE. The ZP values obtained from the unloaded and loaded formulations showed a negative charge. Table 2. Characterization of nanoemulsions by average hydrodynamic diameter (DS; nm), Polydispersity Index (PDI) and Zeta Potential (Zeta, mV) values. NE NE+Mo NE+Ac-Mo NE+Sr-Mo DS (nm) 123.09 ± 1.15 181.3 ± 1.872 134.8 ± 0.763 170.3 ± 0.721 PDI 0.369 ± 0.2 0.086 ± 0.031 0.208 ± 0.011 0.086 ± 0.020 Zeta (mV) -17.4 ± 0.10 -31.7 ± 1.32 -20.9 ± 0.321 -15.3 ± 0.794 Mo – morin; Ac-Mo – acetylated morin; Sr-Mo– morin complexed with strontium. Scanning electron microscopy images from multispecies biofilms treated with NE containing or not Mo and its derivatives are presented in Figures 2A to 2E . Evident biofilm disorganization and areas of “crack” with substantial reduction of bacterial presence and extracellular matrix can be observed in Figures 2A, 2B and 2C when specimens were treated with NE+Mo, NE+Ac-Mo and NE+Sr-Mo, respectively. NE did not affect the biofilm organization and structure ( Figure 2D) , similar to the control group ( Figure 2E ). All NE containing Mo or its derivatives significantly reduced bacterial counts compared to NE and the control group (culture media), as observed in Figure 2F . However, the antibiofilm effect was higher in specimens treated with NE+Ac-Mo and its effect was superior to CHX. Figure 3 shows the effect of 24h treatment with NE+Mo, NE+Ac-Mo and NE+Sr-Mo on 2-weeks multispecies biofilms formed inside human root canals. Representative confocal images of each group are observed in Figures 3A-E showing the dead (red spots) and live (green spots) patterns among the groups. The highest reduction was observed for specimens treated with NE+Ac-Mo (75.87%) followed by Ne+Sr-Mo (59.47%), CHX (50.1%) and Mo (46.17%), with no difference between the last three groups. NE had no antibiofilm effect with minimal reduction like observed in the control group (about 5%). Figure 4 shows the effect of 24h treatment with NE extract dilutions (with Mo and its derivates and controls) on fibroblast cells after 24h exposure. All NE (with or without antimicrobials) at 25 and 50% presented cytotoxic effect, differing from the control group, and reducing cell viability below 30%. At 12.5% dilution were cytocompatible, however NE+Sr-Mo differed from NE+Mo and NE+Ac-Mo and from the control group, but cell metabolism was higher than 80%. Discussion New therapeutic approaches combining antimicrobial substances and drug delivery systems can be promising strategies to effectively eliminate residual bacteria after chemical-mechanical treatment, considering that their persistence may cause periapical lesions. In this study, morin and its derivatives, alone or loaded in nanoemulsions, were effective against oral bacteria and cytocompatibility at lower concentrations, so the null hypotheses were rejected. Morin has been proven to be a potential antimicrobial candidate because of their broad effect against different pathogenic microorganisms, including bacteria, fungi, and viruses [ 18 , 38 , 39 ]. In the present study, morin had bactericidal action, presenting MIC/MBC values from 0.125 to 1 mg/mL. In a previous study, among nine flavonoids tested, morin was the most effective antimicrobial agent, inhibiting the following oral pathogenic microorganisms: Agreggatibacter actinomycetemcomitans, Actinomyces naeslundii, A. viscosus, E. faecalis, Escherichia coli, L. casei, Staphylococcus aureus and C. albicans from concentrations 0.69 to 1.03 mg/mL by Kirby-Bauer disc diffusion method [ 18 ]. Studies have appointed possible mechanisms for morin antibacterial action, such as the destabilization of the bacterial membrane by disordering and disorientation of the membrane lipids and induced leakage from the vesicle; inhibition of helicases, which are essential for DNA replication) Moreover, inhibition of F1F0ATPase controls ATP hydrolysis and synthesis [ 40 – 42 ]. Here, morin derivatives Ac-Mo and Sr-Mo also had an antibacterial effect. However, MIC/MBC values for Ac-Mo were higher than those determined for Mo when tested on S. mutans , L. casei and F. nucleatum . Chemically, the five hydroxyl groups of Mo were replaced by five methyl groups to generate Ac-Mo, aiming to increase the bioactivity and bioavailability of the compound for clinical application [ 29 ]. Previous studies showed that acetylation of curcumin and quercetin has increased the antimicrobial and antiviral activity of the natural compounds [ 29 , 43 ]. For Sr-Mo, the site for Sr complexation was between 3-OH and 4C = O groups presented in the C ring of morin [ 22 ], one of the preferred metal binding sites for flavonoid chelation. Natural derivatives from morin, morin-3-O-α-L-lyxopyranoside and morin-3-O-α-L arabopyranoside from guava leaves ( Psidium guajava L .) have shown antibacterial effect with inhibitory concentrations of 300 µg/mL for Bacillus cereus and 150 µg/ml for Salmonella enteritidis , respectively [ 44 ]. Two sulfonic derivatives of morin were synthesized and showed better inhibitory effects against S. aureus ATCC 29213 than morin, with MIC = 3.9 µg/mL [ 20 ]. Morin has also been complexed with ions, such as lanthanum (III), gadolinium (III) and lutetium (III) and demonstrated inhibitory action against E. coli, Klebsiella pneumoniae, S. aureus at different concentrations [ 21 ]. No study evaluated the antimicrobial effect of acetylated morin or morin complexed with strontium. Although the mechanism of flavonoid–metal complexes has not yet been defined, chemical modifications on flavonoid structure will probably change the intracellular targets to be different from the original compound, and the effect of the metal ions against bacteria should be considered [ 45 ]. In the current study, morin and both morin derivatives (Ac-Mo and Sr-Mo), when loaded in nanoemulsions (NE), were effective against multispecies biofilms, significantly reducing the bacterial counts in microplates and inside dentin tubules. For both assays, Ne + Ac-Mo presented a higher effect on multispecies biofilms than NE + Mo, NE + Sr-Mo and CHX. Morin has shown the ability to inhibit the formation of initial biofilms and reduce the virulence factors, such as adhesion and aggregation synthesis of different microorganisms [ 19 , 46 – 48 ]. When incorporated in alginate/gellan gum films and tablets, morin also reduced the acidogenicity, microbial viability and EPS concentrations in S. mutans and A. naeslundii monospecies biofilms and polymicrobial biofilms [ 27 , 28 ]. Considering the results and observing the SEM images obtained from this study, NEs loaded with morin, especially NE + Ac-MO, could disrupt the biofilms and substantially reduce the bacterial amounts. Studies with morin-loaded NE evaluated their pharmacological properties [ 49 , 50 ], and no study was found about the antimicrobial/antibiofilm activity of those NEs. Other studies showed the sustainable antimicrobial effect of nanoemulsions containing polyphenols and the ability to inhibit pathogenic bacterial species, such as S. aureus, Salmonellla typhimurium, E. coli, S. enteritidis, Listeria monocytogenes [ 26 , 51 – 53 ]. The physicochemical characterization of NE in this study showed an average hydrodynamic diameter between 123 and 170 nm and PD indices from 0.086 to 0.369, similar to those found by Jangid and Jain [ 49 ] for similar NE containing morin, indicating a good droplet size distribution (in nanoscale) and stability of the NE system. For NE + Ac-Mo and NE + Sr-Mo, PDI was lower than the other NE and less than 0.25, a value considered ideal for reducing the instability phenomena caused by droplet kinetics over time [ 54 ]. Zeta potential (ZP) is a valuable tool for estimating the stability of emulsions because it determines the electrostatic repulsion between globules. In this study, the ZP values obtained from the unloaded and loaded formulations showed a negative charge below − 31mV and probably was influenced by the surfactant. For the ZP, ideal values for the prevalence of electrostatic repulsion are considered when the electrical charge of the drop is above + 30 mV or below − 30 mV [ 54 ]. Duarte et al. [ 55 ] developed characterized nanoemulsions (loaded with cymene and myrcene) using the same methodology proposed in the present study and observed PS from 98 to 118nm, PDI from 0.209-to 0.246 and zeta potential from − 25 to -20mV over 60 days demonstrated NE stability. They also tested compounds alone, loaded them in NE, and noted that the NE showed controlled release properties. The metabolic activity (%) of fibroblasts (NHI/3T3) was above 70% after 24h of exposure to morin and derivatives at 0.25 mg/mL, being considered cytocompatible (Mo: 79.14%; Ac-Mo: 119.34% and Sr-Mo: 94.59%). All Mo derivatives had better results compared to CHX, which was highly cytotoxic at the concentrations tested. When NE was diluted to 12.5% dilution, all NE showed cytocompatibility, and NE + Mo and NE + Ac-Mo increased the metabolic activity in comparison to NE and NE + Sr-Mo, with no statistical difference from the control group. In vitro and In vivo studies have demonstrated that morin exhibits low cytotoxicity ( IC50 = 250 ± 40 µM in human leucocytes), and its chronic administration is well tolerated considering that 300mg/Kg as the maximum dose without causing adverse effects in humans [ 27 , 56 – 58 ]. NIH-3T3 fibroblasts were treated with 1% naringin NE, and cell viability was 88.74%, 86.12% and 83.56% when the NE was diluted, and naringin was at 0.25, 0.75 and 1.25 µM concentrations [ 59 ]. Considering the inherent limitations of the in vitro study, morin and its derivatives, free in solution or loaded in nanoemulsions, were effective against multispecies biofilms and cytocompatible at lower concentrations. Chemical acetylation of morin significantly increased its antimicrobial/antibiofilm properties and could be used as an intracanal medication. Declarations Data availability All data is provided within the manuscript. Conflict of interests The authors declare no competing interests. Ethical Approval This study was approved by Ethical Committee in Human Research of the Araçatuba School of Dentistry, Araçatuba, (CAAE - 38042320.6.0000.5420). Financial support This work was financially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, financial code #001), Universidade Estadual Paulista (UNESP, #013/2022-PROPG, PIBIC-REITORIA 3648), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, #19/25125-7), Fundação para a Ciência e a Tecnologia – FCT, Portugal (UIDP/04279/2020). Acknowledgements The author would like to thank Elton José de Souza from Ilha Solteira School of Engineering – UNESP and Marcia Graeff for Bauru School of Dentistry – USP for scanning electron microscopy and confocal microscopy support, respectively. Contributions CRediT (Contribuidor Roles Taxonomy) Conceptualization: [Jesse Augusto Pereira, Aimée Maria Guiotti, Jonas Lobato Duarte, Ana Paula Ramos, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Methodology: [Jesse Augusto Pereira, Rafael Araújo Rios, Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Ana Paula Ramos, Cristiane Duque]; Formal analysis: [Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Cristiane Duque]; Funding acquisition: [Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Project administration: [Ana Paula Ramos, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Supervision: [Aimée Maria Guiotti, Ana Paula Ramos, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Validation: [Ana Paula Ramos, Anil Kishen, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Visualization: [Jesse Augusto Pereira, Rafael Araújo Rios, Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Ana Paula Ramos, Aimée Maria Guiotti, Anil Kishen, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Writing–Original Draft: [Jesse Augusto Pereira, Jonas Lobato Duarte, Cristiane Duque]; Writing–Review & Editing: [Jesse Augusto Pereira, Rafael Araújo Rios, Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Ana Paula Ramo, Aimée Maria Guiotti, Anil Kishen, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque] References Hargreaves KM, Geisler T, Henry M, Wang Y (2008) Regeneration potential of the young permanent tooth: what does the future hold? 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Curr Med Chem 23:774-791. https://10.2174/0929867323666160106150821 Akrawi SH, Gorain B, Nair AB, Choudhury H, Pandey M, Shah JN, Venugopala KN (2020) Development and optimization of naringenin-loaded chitosan-coated nanoemulsion for topical therapy in wound healing. Pharmaceutics 12:893. https://10.3390/pharmaceutics12090893. Additional Declarations No competing interests reported. Supplementary Files SupplFigureTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5923566","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408678038,"identity":"4a89be10-112c-40cd-8292-c9154f689a74","order_by":0,"name":"Jesse Augusto Pereira","email":"","orcid":"","institution":"Araçatuba Dental School, São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Jesse","middleName":"Augusto","lastName":"Pereira","suffix":""},{"id":408678039,"identity":"2b78a762-72f9-4629-80fb-104cdb51164d","order_by":1,"name":"Rafael Araújo Rios","email":"","orcid":"","institution":"Araçatuba Dental School, São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"Araújo","lastName":"Rios","suffix":""},{"id":408678040,"identity":"5330c543-15ed-45ea-9447-f1f2dcf0c44a","order_by":2,"name":"Jonatas Lobato Duarte","email":"","orcid":"","institution":"São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Jonatas","middleName":"Lobato","lastName":"Duarte","suffix":""},{"id":408678041,"identity":"fe591eb1-9d0e-4c17-893f-2e6f87929ae0","order_by":3,"name":"Graciele Ribeiro Moraes","email":"","orcid":"","institution":"São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Graciele","middleName":"Ribeiro","lastName":"Moraes","suffix":""},{"id":408678042,"identity":"e68f39ed-a968-47f0-aa7e-bf2f374c3242","order_by":4,"name":"Ana Paula Ramos","email":"","orcid":"","institution":"University of São Paulo (USP)","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Paula","lastName":"Ramos","suffix":""},{"id":408678043,"identity":"9f78cc26-8b0b-45f2-9aa8-a2bfa8f41ba2","order_by":5,"name":"Aimée Maria Guiotti","email":"","orcid":"","institution":"Araçatuba Dental School, São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Aimée","middleName":"Maria","lastName":"Guiotti","suffix":""},{"id":408678044,"identity":"fc583881-df41-4a8e-b030-1961b256a18e","order_by":6,"name":"Anil Kishen","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Anil","middleName":"","lastName":"Kishen","suffix":""},{"id":408678045,"identity":"850c829c-d3e2-4913-a94d-910047ce837e","order_by":7,"name":"Luís Octavio Regasini","email":"","orcid":"","institution":"São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Luís","middleName":"Octavio","lastName":"Regasini","suffix":""},{"id":408678046,"identity":"924a0449-dff9-4936-b56e-9e1e6df3e11a","order_by":8,"name":"Marlus Chorilli","email":"","orcid":"","institution":"São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Marlus","middleName":"","lastName":"Chorilli","suffix":""},{"id":408678047,"identity":"669d2db3-d42a-4fcd-a0ad-6130c9e9c476","order_by":9,"name":"Cristiane Duque","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAmElEQVRIiWNgGAWjYFCCBBBhQ7qWNAj7AAlaDpOghb89x/A2T8X5xP7ZDWyPPxCjReLMG2NrnjO3E2fcOcBuQJQtBhI5ZtK8bbcTN0gksEmQoOXfOZK1NBwgQYvEmWfFlnOOJRvPuHOw3eAMMVr425M33nhTYyfbP7v52IMKYrSAbYKQjG3EaoBrYWAjXssoGAWjYBSMKAAA7DEyw943QYUAAAAASUVORK5CYII=","orcid":"","institution":"Araçatuba Dental School, São Paulo State University (UNESP)","correspondingAuthor":true,"prefix":"","firstName":"Cristiane","middleName":"","lastName":"Duque","suffix":""}],"badges":[],"createdAt":"2025-01-29 10:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5923566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5923566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75187951,"identity":"a93aaf28-7489-483b-9a1b-b9d7479230db","added_by":"auto","created_at":"2025-01-31 17:59:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25727,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic activity (means/SD) of fibroblasts after 24h treatment with serial dilutions of the compounds Mo, Ac-Mo, Sr-Mo. Concentrations in mg/mL. Mo – morin; Ac-MO – acetylated morin; Sr-MO– morin complexed with strontium. Control: DMEM set as 100%.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e* \u003c/sup\u003eStatistically different from other groups, according to ANOVA and Tukey tests (p\u0026lt;0.05) \u003csup\u003e# \u003c/sup\u003eStatistically different from control group, according to ANOVA and Tukey tests (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5923566/v1/d786110df443c7accdd126dd.png"},{"id":75187989,"identity":"7e9b1ce4-5722-4eaa-b75a-6eae27f7bca0","added_by":"auto","created_at":"2025-01-31 17:59:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":268008,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative scanning electron microscopy (SEM) images of 7-days multispecies biofilms under 1000x magnification. Biofilms were treated for 24h with A - NE+Mo 2mg/mL; B – NE+ Ac-Mo 2mg/mL, C – NE + Sr-Mo 2mg/mL, D – CHX at 0.5 mg/mL; E - NE (with no compounds), F - Control – Bacterial growth without antimicrobial agents. G. Means (SD) of the bacterial counts detected after 24h of the biofilm treatment with nanoemulsions and control. NE – nanoemulsion; Mo – morin; Ac-MO – acetylated morin; Sr-MO– morin complexed with strontium.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e* \u003c/sup\u003eStatistically different from other groups, according to ANOVA and Tukey tests (p\u0026lt;0.05)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e# \u003c/sup\u003eStatistically different from control group, according to ANOVA and Tukey tests (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5923566/v1/bf6daa34081abc8c80492c8b.png"},{"id":75187954,"identity":"7cd098dd-e9d9-44b0-a46e-5c12ba788117","added_by":"auto","created_at":"2025-01-31 17:59:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":401011,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal microscopy images of bovine root dentin specimens contaminated for 14 days with multispecies biofilms and treated for 24 h with the following groups: A - NE+Mo 2mg/mL; B – NE+ Ac-Mo 2mg/mL, C – NE + Sr-Mo 2mg/mL, D – CHX at 0.5 mg/mL; E - NE (with no compounds), F - Control – Bacterial growth without antimicrobial agents and G - Mean (SD) of the percentages of dead cells from multispecies biofilms formed in root dentin of bovine teeth, after 24h of treatment with nanoemulsions and controls.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e* \u003c/sup\u003eStatistically different from the other groups, according to ANOVA and Tukey tests (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e# \u003c/sup\u003eStatistically different from the control group, according to ANOVA and Tukey tests (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5923566/v1/034de17b463382b835618921.png"},{"id":75187955,"identity":"8bc1ffe3-8d19-45d0-8aea-299affe7760c","added_by":"auto","created_at":"2025-01-31 17:59:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16183,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic activity (means/SD) of fibroblasts after 24h treatment with serial dilutions of the extract of nanoemulsions NE+Mo, NE+Ac-Mo, NE+Sr-Mo. Mo – morin; Ac-Mo – acetylated morin; Sr-Mo– morin complexed with strontium. Control: DMEM set as 100%.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e* \u003c/sup\u003eStatistically different from the other groups, according to ANOVA and Tukey tests (p\u0026lt;0.05)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e# \u003c/sup\u003eStatistically different from the control group, according to ANOVA and Tukey tests (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5923566/v1/3f4020cc01aa1a7e1eb94e7b.png"},{"id":75301562,"identity":"b0bc6e40-2b0f-4b1e-8274-b643b2936d5c","added_by":"auto","created_at":"2025-02-03 07:39:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1783783,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5923566/v1/c3ed1f5b-8a9e-46b7-8012-c817790bb1d4.pdf"},{"id":75187981,"identity":"62c5bbe7-c244-4d0e-a54e-555b14cb57f2","added_by":"auto","created_at":"2025-01-31 17:59:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":376063,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigureTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5923566/v1/c51e3d86504c448aa9dc8ef6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eClinical Oral Investigations Biological and Physicochemical Characterization of Nanoemulsions Containing Morin and its Derivatives for Endodontic Purposes\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTreating young permanent teeth with irreversible pulp damage is a significant challenge for endodontists and pediatric dentists. Since the root canals and apices of the permanent teeth are still not completed, these processes can be interrupted due to trauma or infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The conventional treatment, or apexification, involves the removal of vital or necrotic pulp, disinfection, and induction of a calcified barrier in the roots by using calcium hydroxide as an intracanal medication. However, this procedure does not increase the root length and strength, leading to a higher risk of reinfection and tooth fracture [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. New approaches of regenerative endodontics for immature teeth have included minimal or no instrumentation of the dentinal walls and disinfection with effective but non-toxic irrigates/intracanal medicaments to solve the signs and symptoms of pulpal or periapical diseases and allow tissue regeneration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe irrigation solution most used for root canal disinfection is sodium hypochlorite (NaOCl), with concentrations varying from 0.5 to 6%, for its high antimicrobial efficacy and tissue dissolution capacity [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. NaOCl in concentrations from 1 to 5.25% were similarly effective in reducing \u003cem\u003eEnterococcus faecalis\u003c/em\u003e in root canals since large quantities and regular irrigant exchanges are maintained, especially for lower concentrations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Considering the cytotoxicity, NaOCl from 0.5 to 3% reduced 37% of stem cells of the apical papilla (SCAP) survival in an organotypic root canal model; however, after final irrigation with 17% EDTA and saline solution, cell viability was recovered [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Another important antimicrobial agent in endodontics is chlorhexidine, a bisbiguanide with a wide-range effect against Gram-positive and Gram-negative bacteria, fungi, and viruses [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Some studies have demonstrated a higher effect of chlorhexidine in eliminating \u003cem\u003eE. faecalis\u003c/em\u003e from dentin tubules than other medicaments [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Conversely, chlorhexidine has presented cytotoxicity when in contact with odontoblastic cells, even at low concentrations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eManual syringe irrigation is still the most popular method; however, it does not remove debris from secondary canals and apical irregularities. The use of auxiliary means to increase irrigation efficacy, such as passive ultrasonic activation (PUI), can increase the removal of debris, organic tissues, and microorganisms from the root canals. PUI consists of transmitting acoustic energy from an oscillating file or smooth wire to an irrigant solution in the root canal, facilitating the penetration into unassessed root canal areas [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have shown that PUI was more effective in removing dentin and pulp remnants and reducing planktonic bacteria content than conventional irrigation [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA natural polyphenol called morin (3, 5, 7, 20, 40-pentahydroxyflavone) is found in plants such as \u003cem\u003ePrunus dulcis\u003c/em\u003e (almonds), \u003cem\u003eMaclura tinctoria\u003c/em\u003e (figs) and other plants in the Morceau family, such as \u003cem\u003ePsidium guajava\u003c/em\u003e (Indian guava) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Morin has demonstrated an inhibitory effect against several oral pathogenic microorganisms [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and reduces the ability of adhesion and biofilm formation by \u003cem\u003eStreptococcus mutans\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Some studies have synthesized morin derivatives, searching for new compounds with better antimicrobial activity than morin. Sodium salt of morin-5'-sulfonic acid showed an effect against antibiotic-resistant bacteria and clinical isolates of S. aureus with lower minimal inhibitory concentration (MIC) values than morin but higher MIC values against \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strains [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Morin has also been complexed with ions, such as lanthanum (III), gadolinium (III) and lutetium (III) and demonstrated inhibitory action against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e at different concentrations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Morin complexed with strontium (Sr\u0026thinsp;+\u0026thinsp;2) reduced the differentiation of pre-osteoclasts and maintained the mineralization ability of osteoblasts compared to noncomplexed morin and strontium ranelate at the same concentrations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe administration of phytomedicines generally presents limitations relative to their low aqueous solubility, low permeability, high molecular weight, enzymatic degradation, low bioavailability, and drug stability [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Nanoemulsions are considered a high-standard topical drug delivery by controlling drug release and focusing on site-specific action [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Nanoemulsions are oil-in-water (o/w) or water-in-oil (w/o) dispersions of two immiscible liquids that have been stabilized using a suitable surfactant. The following characteristics are mandatory to develop nanoemulsions: droplet size of 1-100 nm; mixing two fluids with surfactant; protection of medicines from physical and chemical degradation; being non-toxic; increasing the solubility of medicines; and facilitating the distribution of many medicines [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Cinnamic acid-loaded nanoemulsion was compared to pure cinnamic acid. It showed a higher effect in inhibiting planktonic cells and biofilms of \u003cem\u003eS. typhimurium\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e due to the subcellular size of nanoemulsion that allowed it to reach bacterial targets effectively [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Some studies have demonstrated that morin in muco-adhesive controlled-release systems (gellan gun-based) effectively controlled single-species and multispecies biofilms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the limited research on new compounds that have a wide range of therapeutic effects and drug delivery systems for endodontic applications, especially for treating immature teeth, this study aimed to achieve two main objectives. First, the study sought to synthesize two morin derivatives and test their antimicrobial and cytotoxic effects. Second, the study aimed to develop and characterize nanoemulsions containing morin and two morin derivatives, and then evaluate their cytotoxicity and effect on multispecies biofilms. The null hypothesis was that morin derivatives alone or loaded in nanoemulsions did not exhibit antimicrobial or antibiofilm activity and cytocompatibility.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eUnless otherwise specified, compounds and reagents were purchased from Sigma-Aldrich® (St. Louis, MO, USA), bacterial culture media from Difco (Kansas City, MO, USA) and culture media/supplements for cell cultures from ThermoFisher Scientific® (Waltham, MA, USA).\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eSynthesis of penta-acetylated morin (Ac-Mo)\u003c/h2\u003e\n \u003cp\u003eAn organic synthesis reaction for morin acetylation was carried out as previously described in Guimarães et al. [29] with some modifications. Briefly, morin hydrate (500mg, Sigma #M4008) was dissolved in 20mL acetic anhydride and 20mL pyridine in a round bottom flask and was heated between 94°C and 100°C in an oil bath. The reaction was maintained for 48h and monitored by analytical thin-layer chromatography (TLC) plates, using ultraviolet light (at 254 and 365 nm) and in the chemical agent sulfuric anisaldehyde. After the conversion of morin, the mixture was poured onto a beaker with crushed ice and stored in a refrigerator for 24 hours. The precipitate was vacuum filtered and left to dry at room temperature, presenting as a brown powder. For purification, the crude product (0.628mg) was subjected to a chromatography column over silica gel, using a mixture of the solvent's hexane and ethyl acetate in a 60:40 ratio, yielded around 56 fractions. These fractions were subjected to analytical thin-layer chromatography for a first purification analysis. The purification process by column chromatography was repeated, yielding around 145 fractions subjected to analytical thin-layer chromatography. 1H-NMR and 13C-NMR spectra were obtained on a Bruker Avance III HD (Bruker, Germany) at 400 MHz.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSynthesis of strontium-morin complex (Sr-Mo)\u003c/h3\u003e\n\u003cp\u003eThe synthesis of Sr-Mo was performed according to Cruz et al. [22]. Morin hydrate (0.150g) was solubilized in 50.0 mL methanol with 100 mL of NH4OH in a round bottom flask under continuous stirring. After that, SrCl2.6H2O (0.399g) was slowly added, and the mixture was kept under agitation for 2 h. Then, the product was centrifuged, washed with chloroform: t-butanol solution (1: 1, v/v) and dried at 37oC. The complete structural characterization of the final product is described elsewhere [22].\u003c/p\u003e\n\u003ch3\u003eStock solutions and controls\u003c/h3\u003e\n\u003cp\u003eStock solutions of morin and their derivatives were prepared and frozen in dimethyl sulfoxide – DMSO at 30mg/mL. Only work solutions (in culture medium) containing up to 1% DMSO were used for the subsequent tests so as not to influence the results. For microbiological and cytotoxicity assays, chlorhexidine digluconate (Farmácia Manipullis, Araçatuba – SP/BR) in deionized water was chosen as a positive control (antimicrobial agent considered as a gold standard in Dentistry), and nanoemulsion with no compounds (NE) and culture media (Control) were determined as controls. All compounds were sterilized by filtration through a 0.22 µm membrane filter. \u003cstrong\u003eSuppl. Table\u0026nbsp;1\u003c/strong\u003e presents the chemical structure, empirical formula, and molecular weight of morin and its derivatives. Unless otherwise mentioned, the assays were performed in triplicate in three independent days (n = 9).\u003c/p\u003e\n\u003ch3\u003eDetermination of antimicrobial activity and cytotoxicity of compounds\u003c/h3\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003eStandard strains and growth conditions\u003c/h2\u003e\n \u003cp\u003eThe following bacterial strains were donated by Oswaldo Cruz Foundation (FIOCRUZ, RJ, Brazil) for this study: \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (ATCC 51299), \u003cem\u003eActinomyces israelii\u003c/em\u003e (ATCC 12102), \u003cem\u003eStreptococcus mutans\u003c/em\u003e (ATCC 25175), \u003cem\u003eLactobacillus casei\u003c/em\u003e (ATCC 393), and \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e (ATCC 25586). The culture media used for the growth of each bacterial species were as follows: Mitis Agar Salivarius Agar with 0.2 U/mL bacitracin for \u003cem\u003eS. mutans\u003c/em\u003e, MRS Rogosa Agar for \u003cem\u003eL. casei\u003c/em\u003e, Brain Heart Infusion Agar – BHIA for \u003cem\u003eA. israelii\u003c/em\u003e and \u003cem\u003eE. faecalis. F. nucleatum\u003c/em\u003e was grown in BHIA with 5mg/L of hemin, 5mg/mL of menadione and 5% of defibrinated sheep blood. All plates were incubated at 37°C under anaerobic conditions (AnaeroGen; Oxoid, Hampshire, UK).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eDetermination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)\u003c/h2\u003e\n \u003cp\u003eThe MIC assays were measured using the microdilution method, based on the CLSI standards [30], with some modifications [31]. Briefly, cultures of the bacterial species mentioned previously were subcultured in Miller Hinton (MH) broth medium for 24h at 37° C. Then cells were adjusted to obtain 1-5x 105 cells/mL in MH. Counts were validated by counting on MH agar. The inoculum of each bacterium was then incubated in a serial dilution (from 1 to 0.0001 mg/mL) of each compound - Mo, Ac-Mo and Sr-Mo and controls dissolved in sterilized water. After incubation for 24 h (and 48h for \u003cem\u003eF. nucleatum\u003c/em\u003e) at 37°C, the microtiter plates were stained with resazurin solution at 5 µg/mL for 3h and analyzed at 570 and 600 nm in a spectrophotometer (Biotek, Winooski, VT). Bacterial suspensions were incubated with chlorhexidine (CHX) or without any antimicrobial agent as positive and negative controls, respectively. The MIC was defined as the lowest concentration of the compound in which there was no detectable growth. Media containing the MIC (and two higher concentrations) were serially diluted, plated on MH agar, and incubated at 37°C for 48h. The number of colony-forming units/ml (CFU/mL) was determined by a binocular stereomicroscope. MBC was considered when the compound killed more than 99.9% of the bacteria.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCytotoxicity of the compounds\u003c/h3\u003e\n\u003cp\u003eMetabolic activity was determined on culture of murine fibroblasts L929 fibroblasts, according to Duque et al. [32]. Cells were grown in Dulbecco's modified Eagle medium (DMEM, Dulbecco's Modified Eagle's) supplemented with 10% fetal bovine serum and 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine containing 5% CO2 and 95% air at 37°C until reaching 80% confluence. Then, cells were cultured at a density of 5x 104 cells/well in 96-well plates and incubated for 24 h. The cells were treated with serial dilutions (from 1 to 0.0019 mg/mL) of Mo, Ac-Mo, Sr-Mo and CHX for 24 h. Cells were washed with PBS, and resazurin 70µM in DMEM was added for 4h. After that, the plates were analyzed in a spectrophotometer at 570 and 600nm. The final values were obtained by the subtractions between the absorbance values at both wavelengths. They were converted into a percentage of cell viability, considering the growth in the DMEM medium to be 100% and the means determined for each group.\u003c/p\u003e\n\u003ch3\u003ePreparation and characterization of nanoemulsions\u003c/h3\u003e\n\u003cp\u003eThe preparation of nanoemulsions (NE) was performed according to Bouchemal et al.; 2004 with some modifications [33]. The oil phase composed of medium-chain triglycerides (5% w/w) was weighed together with each compound (Mo, Ac-Mo and Sr-Mo) at 2 mg/mL and polysorbate 20 (5% w/w) and kept under agitation in a magnetic stirrer overnight. The aqueous phase was composed of water q.s.p. (10 g), which was added dropwise onto the oil phase, thus forming a pre-emulsion. This mixture was sonicated for 5 minutes, with a pulse of 1 min, pulse of 30, and amplitude of 27. The formulations were kept at room temperature and evaluated visually after 24h. Then, they were diluted in deionized water (1:25), and NE's average hydrodynamic diameter and polydispersity index (PDI) were determined using dynamic light scattering. Zeta potential was determined by electrophoretic mobility in a Zetasizer 3000 HSA (Malvern Instruments) device, using a 10mW HeNe laser at 633nm (173o of detection angle) at 25\u003csup\u003eo\u003c/sup\u003eC in automatic mode. NEs were kept in the refrigerator at 4 C until used.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eBiological characterization of nanoemulsions\u003c/h2\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eEffect of NEs on multispecies biofilms and scanning electron microscopy\u003c/h2\u003e\n \u003cp\u003eThe multispecies biofilm assays were conducted by mixing \u003cem\u003eE. faecalis\u003c/em\u003e, \u003cem\u003eA. israelii\u003c/em\u003e, \u003cem\u003eL. casei\u003c/em\u003e, \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eF. nucleatum\u003c/em\u003e in equal aliquots in BHI broth containing 1% glucose [31]. Briefly, sterile 96-well U-bottom polystyrene microplates were pre-treated with 150 µL of artificial saliva (800mL of deionized water, 1.6 g of yeast extract, 4 g of peptone, 0.28 g of NaCl, 4 g of sucrose, 0.16 g of CaCl2, 0.16 g of KCl and 0.8 g of mucin) for a period of 4 h at 37°C in the 5% CO₂ (coating phase) [34]. Bacterial cultures were grown individually for 24h; then, they were centrifuged and washed in PB buffer, and the cell number was adjusted to 1-5x103 CFU/mL in BHI broth containing 1% glucose. Then, they were mixed in equal aliquots and inoculated in the microplates. After 7 days of growth in anaerobic conditions, biofilms were washed twice with sterile saline solution, and 100 µl of NE, NE + Mo, NE + Ac-Mo, NE + Sr-Mo and CHX at 0.5 mg/mL (100x the highest MIC) was inserted into each well (n = 6). The plates were incubated for 24h at 37°C in anaerobic conditions. After scraping of biofilms, aliquots from all wells were resuspended, serially diluted, and plated on BHI agar. The plates were incubated for 48h for further counting of CFU/mL [31]. The same experiments were conducted in parallel in coverslips for scanning electron microscopic (SEM) analysis. The samples were dehydrated by washing in a series of ethanol (70% for 10min, 95% for 10 minutes, and 100% for 20 minutes) and air-dried in a desiccator. Afterwards, coverslips were mounted into aluminum stubs, sputter coated with gold, and analyzed in a scanning electron microscope (Leo, Cambridge, MA, USA) [35].\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eEffect of NEs on the multispecies biofilms formed on human root dentin and analysis by Confocal Microscopy\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eSpecimens' preparation and dentin contamination\u003c/h2\u003e\n \u003cp\u003eThis methodology was carried out according to Andrade et al. [36] with some modifications. Human lower permanent incisors were stored in saline solution after cleaning and disinfection in 1% sodium hypochlorite solution for 48h (Ethical committee approval: CAAE − 38042320.6.0000.5420). The crowns and apices were removed, and the length of the roots was standardized at 12mm from apex to coronal edge using a 0.6 mm diamond disk (Isomet 5000; Buehler Ltd, LakeBluff, IL) at 1000 rpm, under irrigation with water. Root canals were assessed with a #6 spherical bur and instrumented with reciprocating files (Reciproc R25; VDW, Munich, Germany), considering the total canal length under irrigation with saline solution. Afterwards, specimens were immersed in tubes with 17% EDTA for 3 min, followed by 5 min in deionized water in an ultrasonic bath. The specimens were sterilized in an autoclave at 121°C for 15 min. Then, they were inserted into a tube containing 1mL BHI (w/hemin and w/menadione) with the canal side up and kept in an ultrasonic bath for 3 min to allow the culture medium to enter the dentinal tubules. Dentin specimens were contaminated by mixing \u003cem\u003eE. faecalis\u003c/em\u003e, \u003cem\u003eA. israelii\u003c/em\u003e, \u003cem\u003eL. casei\u003c/em\u003e, \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eF. nucleatum\u003c/em\u003e in equal aliquots in BHI broth containing 1% glucose, as mentioned before. The tubes were centrifuged at 1400xg, 2000 xg, 3600 xg and 5600 xg in this sequence, twice each, for 5 minutes. A fresh aliquot of bacterial cultures was added between each centrifugation, and the old one was discarded. All microtubes were incubated at 37°C in BHI broth for 14 days under anaerobic conditions to form multispecies biofilms inside dentin tubules. The medium was changed every 72 hours. The dentin specimens were removed from the microtube and washed with sterile water for 1 min. The external surfaces (cementum side) of the specimens were closed with nail polish.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eProtocols of canal treatment and confocal analysis\u003c/h2\u003e\n \u003cp\u003eAll dentin specimens were fixed with double-sided tape in 24 well microplates, and their canals were irrigated with 1mL of 0.5% sodium hypochlorite for 1min (twice), followed by aspiration and new irrigation with 1mL of 17% EDTA for 2 min and 1mL of saline solution for 2 min. Then, the specimens were randomized and divided into groups: control – saline solution; NE (no antimicrobial agents), NE + Mo, NE + Ac-Mo, NE + Sr-Mo (at 2mg/mL) and CHX at 0.5mg/mL. Root canals were filled with 1mL NE and activated with ultrasonic smooth wire (Irri sonic; Helse Dental Technology, Brazil) placed 1.0 mm short of the working length and activated using 3 cycles of 20 seconds) for 1 min. Specimens were then incubated at 37°C for 24h in sealed microplates to avoid dryness. After that, root canals were dried with paper points and immersed in a solution of 0.9 mM KH2PO4 and 1.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e (pH 7.0) for 1 minute to avoid residual effects of the treatments. Then, the specimens were longitudinally sectioned and stained with the fluorescent LIVE/DEAD Backlight Bacterial Viability stain (Molecular Probes, Eugene, OR). The fluorescence of the stained cells was evaluated in a Leica TCS SPE confocal microscope (Leica Microsystems, Germany), and the 2D images (3 specimens and n = 6 images/per group) acquired by the confocal software using the resolution of 1024x1024 pixels. Emission dead (avital) cells stained in red were visualized at 620nm, and emission vital cells stained in green were visualized at 530nm [37]. The proportion of emission dead cells was determined by counting emission dead cells over total cell counts (vital + dead cells) using the Image J 1.48 program (NIH, Bethesda, MA, USA) [31].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eCytotoxicity of NE\u003c/h2\u003e\n \u003cp\u003eThe cytotoxicity of NEs was determined on the culture of murine fibroblasts L929 fibroblasts, following the methodology previously described (item \u003cem\u003eCytotoxicity of the compounds)\u003c/em\u003e. NE were diluted 10x before being applied on cell cultures. The cells were treated with serial dilutions of NE, NE + Mo, NE + Ac-Mo and NE + Sr-Mo (from 50 to 6.25%) for 24 h. Cells were washed with PBS, and resazurin 70µM in DMEM was added for 4h. After that, the plates were analyzed in a spectrophotometer at 570 and 600nm. The final values were obtained by subtracting the absorbance values at both wavelengths, and they were converted into the percentage of cell viability, considering the growth in the DMEM medium to be 100% and the means determined for each group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was accomplished using JAMOVI software (version 2.5.7). Datasets were assessed for analysis of variance (one-way or two-way ANOVA), followed by Tukey's post hoc test. The significance level was set at p \u0026lt; 0.05. Data were displayed as mean ± standard deviation (SD).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eshows the values of minimal inhibitory and bactericidal concentrations (MIC/MBC) in mg/mL determined for morin (Mo) and their derivatives (Ac-Mo and Sr-Mo) against some oral bacteria. All compounds were bactericidal, presenting values of MIC from 0.125 to 0.5 mg/mL and MBC from 0.25 to 1 mg/mL. Ac-Mo presented values of MIC lower than Mo for \u003cem\u003eE. faecalis, A. israelii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eS. mutans\u003c/em\u003e. The values of MIC/MBC for Sr-Mo were higher than Mo when tested on \u003cem\u003eS. mutans\u0026nbsp;\u003c/em\u003eand \u003cem\u003eF. nucleatum\u003c/em\u003e. CHX had the lowest MIC/MBC values for all bacteria tested.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) in mg/mL for the flavonoids and control chlorhexidine against the oral microorganisms tested.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003efaecalis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eA.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eisraelii\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emutans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCasei\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003enucleatum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMorin (Mo)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.5 (1)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.25 (0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.25 (0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.125 (0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.125 (0.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcetylated Morin\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ac-Mo)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.25 (0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.125 (0.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.125 (0.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.125(0.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.125 (0.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSr\u0026ndash;morin complex\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Sr-Mo)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.5 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.25 (0.5)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.5 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.25 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5 (1)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChlorhexidine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003cp\u003e(0.009)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.0001 (0.004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003cp\u003e(0.001)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003cp\u003e(0.001)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003cp\u003e(0.0006)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*In parentheses - MBC: \u0026gt;99.9% cell reduction. MIC results were based on resazurin staining and MBC results were based on CFU/mL count in Miller Hinton Agar (MHA) medium. The growth of microorganisms without antimicrobials in MHA was considered 100%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn \u003cstrong\u003eSuppl. Figure 1A\u003c/strong\u003e signs 2.34; 2.33; 2.32; 2.19 and 2.17 represent the five resonance signals of the five methyls, thus confirming that morin was penta-acetylated. Likewise, in \u003cstrong\u003eSuppl. Figure 1\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e, resonance signals 21.38; 21.32; 21.23; 21.05 and 20.42 represent the five methyls, thus confirming that the penta-acetylation was successful and confirmed through the two experiments. Using NMR spectra, it was determined that the acetylated morin had a good purity level and could proceed to the testing phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1\u003c/strong\u003e presents the metabolic activity (%) of fibroblasts (NHI/3T3) after 24h treatment with different concentrations of Mo, Ac-Mo, and Sr-Mo. At 1mg and 0.5 mg/mL, metabolic activity varied from 21.94 to 56.07% and from 50.75 to 62.52%, respectively, being statistically higher for Mo derivatives. All groups differed from the control group (DMEM). From 0.25 mg/mL, all compounds were considered as cytocompatible since metabolic activity was higher than 70% (Mo: 79.14%; Ac-Mo: 119.34% and Sr-Mo: 94.59%). At this concentration only Mo and Ac-Mo statistically differed from the control. CHX was cytotoxic at all concentrations tested.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNE+Mo, NE+Sr-and NE+Ac-Mo showed a yellowish, brownish, and white color appearance, similar to compounds free, respectively, as observed in \u003cstrong\u003eSuppl. Figure 2\u003c/strong\u003e. Considering the characterization of nanoemulsions shown in\u003cstrong\u003e\u0026nbsp;Table 2\u003c/strong\u003e, the results for particle size were similar among the groups. PDI is an index that assesses the relative homogeneity in the sizes of particles distributed in samples, and it was calculated by dividing the average droplet size by an average number of measured droplets. Light-scattering analysis showed mean PDI values of 0.086 for NE containing Mo and Sr-Mo after formulation and monomodal distribution. For NE+Ac-Mo and NE-Sr-Mo, PDI was lower than the other NE. This parameter directly reflects droplet size homogeneity in the NE. The ZP values obtained from the unloaded and loaded formulations showed a negative charge.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Characterization of nanoemulsions by average hydrodynamic diameter (DS; nm), Polydispersity Index (PDI) and Zeta Potential (Zeta, mV) values.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNE+Mo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNE+Ac-Mo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNE+Sr-Mo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eDS (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e123.09 \u0026plusmn; 1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e181.3 \u0026plusmn; 1.872\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e134.8 \u0026plusmn; 0.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e170.3 \u0026plusmn; 0.721\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ePDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.369\u0026nbsp;\u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.086 \u0026plusmn; 0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.208 \u0026plusmn; 0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.086 \u0026plusmn; 0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eZeta (mV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e-17.4 \u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e-31.7 \u0026plusmn; 1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e-20.9 \u0026plusmn; 0.321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e-15.3 \u0026plusmn; 0.794\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMo \u0026ndash; morin; Ac-Mo \u0026ndash; acetylated morin; Sr-Mo\u0026ndash; morin complexed with strontium.\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy images from multispecies biofilms treated with NE containing or not Mo and its derivatives are presented in \u003cstrong\u003eFigures 2A to 2E\u003c/strong\u003e. Evident biofilm disorganization and areas of \u0026ldquo;crack\u0026rdquo; with substantial reduction of bacterial presence and extracellular matrix can be observed in Figures \u003cstrong\u003e2A, 2B and 2C\u003c/strong\u003e when specimens were treated with NE+Mo, NE+Ac-Mo and NE+Sr-Mo, respectively. NE did not affect the biofilm organization and structure (\u003cstrong\u003eFigure 2D)\u003c/strong\u003e, similar to the control group (\u003cstrong\u003eFigure 2E\u003c/strong\u003e). All NE containing Mo or its derivatives significantly reduced bacterial counts compared to NE and the control group (culture media), as observed in \u003cstrong\u003eFigure 2F\u003c/strong\u003e. However, the antibiofilm effect was higher in specimens treated with NE+Ac-Mo and its effect was superior to CHX.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3\u0026nbsp;\u003c/strong\u003eshows the effect of 24h treatment with NE+Mo, NE+Ac-Mo and NE+Sr-Mo on 2-weeks multispecies biofilms formed inside human root canals. Representative confocal images of each group are observed in \u003cstrong\u003eFigures 3A-E\u003c/strong\u003e showing the dead (red spots) and live (green spots) patterns among the groups. The highest reduction was observed for specimens treated with NE+Ac-Mo (75.87%) followed by Ne+Sr-Mo (59.47%), CHX (50.1%) and Mo (46.17%), with no difference between the last three groups. NE had no antibiofilm effect with minimal reduction like observed in the control group (about 5%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e shows the effect of 24h treatment with NE extract dilutions (with Mo and its derivates and controls) on fibroblast cells after 24h exposure. All NE (with or without antimicrobials) at 25 and 50% presented cytotoxic effect, differing from the control group, and reducing cell viability below 30%. At 12.5% dilution were cytocompatible, however NE+Sr-Mo differed from NE+Mo and NE+Ac-Mo and from the control group, but cell metabolism was higher than 80%.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNew therapeutic approaches combining antimicrobial substances and drug delivery systems can be promising strategies to effectively eliminate residual bacteria after chemical-mechanical treatment, considering that their persistence may cause periapical lesions. In this study, morin and its derivatives, alone or loaded in nanoemulsions, were effective against oral bacteria and cytocompatibility at lower concentrations, so the null hypotheses were rejected.\u003c/p\u003e \u003cp\u003eMorin has been proven to be a potential antimicrobial candidate because of their broad effect against different pathogenic microorganisms, including bacteria, fungi, and viruses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the present study, morin had bactericidal action, presenting MIC/MBC values from 0.125 to 1 mg/mL. In a previous study, among nine flavonoids tested, morin was the most effective antimicrobial agent, inhibiting the following oral pathogenic microorganisms: \u003cem\u003eAgreggatibacter actinomycetemcomitans, Actinomyces naeslundii, A. viscosus, E. faecalis, Escherichia coli, L. casei, Staphylococcus aureus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e from concentrations 0.69 to 1.03 mg/mL by Kirby-Bauer disc diffusion method [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies have appointed possible mechanisms for morin antibacterial action, such as the destabilization of the bacterial membrane by disordering and disorientation of the membrane lipids and induced leakage from the vesicle; inhibition of helicases, which are essential for DNA replication) Moreover, inhibition of F1F0ATPase controls ATP hydrolysis and synthesis [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, morin derivatives Ac-Mo and Sr-Mo also had an antibacterial effect. However, MIC/MBC values for Ac-Mo were higher than those determined for Mo when tested on \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eL. casei\u003c/em\u003e and \u003cem\u003eF. nucleatum\u003c/em\u003e. Chemically, the five hydroxyl groups of Mo were replaced by five methyl groups to generate Ac-Mo, aiming to increase the bioactivity and bioavailability of the compound for clinical application [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies showed that acetylation of curcumin and quercetin has increased the antimicrobial and antiviral activity of the natural compounds [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For Sr-Mo, the site for Sr complexation was between 3-OH and 4C\u0026thinsp;=\u0026thinsp;O groups presented in the C ring of morin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], one of the preferred metal binding sites for flavonoid chelation. Natural derivatives from morin, morin-3-O-α-L-lyxopyranoside and morin-3-O-α-L arabopyranoside from guava leaves (\u003cem\u003ePsidium guajava L\u003c/em\u003e.) have shown antibacterial effect with inhibitory concentrations of 300 \u0026micro;g/mL for \u003cem\u003eBacillus cereus\u003c/em\u003e and 150 \u0026micro;g/ml for \u003cem\u003eSalmonella enteritidis\u003c/em\u003e, respectively [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Two sulfonic derivatives of morin were synthesized and showed better inhibitory effects against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 than morin, with MIC\u0026thinsp;=\u0026thinsp;3.9 \u0026micro;g/mL [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Morin has also been complexed with ions, such as lanthanum (III), gadolinium (III) and lutetium (III) and demonstrated inhibitory action against \u003cem\u003eE. coli, Klebsiella pneumoniae, S. aureus\u003c/em\u003e at different concentrations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. No study evaluated the antimicrobial effect of acetylated morin or morin complexed with strontium. Although the mechanism of flavonoid\u0026ndash;metal complexes has not yet been defined, chemical modifications on flavonoid structure will probably change the intracellular targets to be different from the original compound, and the effect of the metal ions against bacteria should be considered [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study, morin and both morin derivatives (Ac-Mo and Sr-Mo), when loaded in nanoemulsions (NE), were effective against multispecies biofilms, significantly reducing the bacterial counts in microplates and inside dentin tubules. For both assays, Ne\u0026thinsp;+\u0026thinsp;Ac-Mo presented a higher effect on multispecies biofilms than NE\u0026thinsp;+\u0026thinsp;Mo, NE\u0026thinsp;+\u0026thinsp;Sr-Mo and CHX. Morin has shown the ability to inhibit the formation of initial biofilms and reduce the virulence factors, such as adhesion and aggregation synthesis of different microorganisms [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. When incorporated in alginate/gellan gum films and tablets, morin also reduced the acidogenicity, microbial viability and EPS concentrations in \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eA. naeslundii\u003c/em\u003e monospecies biofilms and polymicrobial biofilms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Considering the results and observing the SEM images obtained from this study, NEs loaded with morin, especially NE\u0026thinsp;+\u0026thinsp;Ac-MO, could disrupt the biofilms and substantially reduce the bacterial amounts. Studies with morin-loaded NE evaluated their pharmacological properties [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and no study was found about the antimicrobial/antibiofilm activity of those NEs. Other studies showed the sustainable antimicrobial effect of nanoemulsions containing polyphenols and the ability to inhibit pathogenic bacterial species, such as \u003cem\u003eS. aureus, Salmonellla typhimurium, E. coli, S. enteritidis, Listeria monocytogenes\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe physicochemical characterization of NE in this study showed an average hydrodynamic diameter between 123 and 170 nm and PD indices from 0.086 to 0.369, similar to those found by Jangid and Jain [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] for similar NE containing morin, indicating a good droplet size distribution (in nanoscale) and stability of the NE system. For NE\u0026thinsp;+\u0026thinsp;Ac-Mo and NE\u0026thinsp;+\u0026thinsp;Sr-Mo, PDI was lower than the other NE and less than 0.25, a value considered ideal for reducing the instability phenomena caused by droplet kinetics over time [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Zeta potential (ZP) is a valuable tool for estimating the stability of emulsions because it determines the electrostatic repulsion between globules. In this study, the ZP values obtained from the unloaded and loaded formulations showed a negative charge below \u0026minus;\u0026thinsp;31mV and probably was influenced by the surfactant. For the ZP, ideal values for the prevalence of electrostatic repulsion are considered when the electrical charge of the drop is above +\u0026thinsp;30 mV or below \u0026minus;\u0026thinsp;30 mV [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Duarte et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] developed characterized nanoemulsions (loaded with cymene and myrcene) using the same methodology proposed in the present study and observed PS from 98 to 118nm, PDI from 0.209-to 0.246 and zeta potential from \u0026minus;\u0026thinsp;25 to -20mV over 60 days demonstrated NE stability. They also tested compounds alone, loaded them in NE, and noted that the NE showed controlled release properties.\u003c/p\u003e \u003cp\u003eThe metabolic activity (%) of fibroblasts (NHI/3T3) was above 70% after 24h of exposure to morin and derivatives at 0.25 mg/mL, being considered cytocompatible (Mo: 79.14%; Ac-Mo: 119.34% and Sr-Mo: 94.59%). All Mo derivatives had better results compared to CHX, which was highly cytotoxic at the concentrations tested. When NE was diluted to 12.5% dilution, all NE showed cytocompatibility, and NE\u0026thinsp;+\u0026thinsp;Mo and NE\u0026thinsp;+\u0026thinsp;Ac-Mo increased the metabolic activity in comparison to NE and NE\u0026thinsp;+\u0026thinsp;Sr-Mo, with no statistical difference from the control group. In vitro and In vivo studies have demonstrated that morin exhibits low cytotoxicity \u003cem\u003e(\u003c/em\u003eIC50\u0026thinsp;=\u0026thinsp;250\u0026thinsp;\u0026plusmn;\u0026thinsp;40 \u0026micro;M in human leucocytes), and its chronic administration is well tolerated considering that 300mg/Kg as the maximum dose without causing adverse effects in humans [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. NIH-3T3 fibroblasts were treated with 1% naringin NE, and cell viability was 88.74%, 86.12% and 83.56% when the NE was diluted, and naringin was at 0.25, 0.75 and 1.25 \u0026micro;M concentrations [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Considering the inherent limitations of the in vitro study, morin and its derivatives, free in solution or loaded in nanoemulsions, were effective against multispecies biofilms and cytocompatible at lower concentrations. Chemical acetylation of morin significantly increased its antimicrobial/antibiofilm properties and could be used as an intracanal medication.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by Ethical Committee in Human Research of the Araçatuba School of Dentistry, Araçatuba, (CAAE - 38042320.6.0000.5420).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eFinancial support\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis work was financially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, financial code #001), Universidade Estadual Paulista (UNESP, #013/2022-PROPG, PIBIC-REITORIA 3648), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, #19/25125-7), Fundação para a Ciência e a Tecnologia – FCT, Portugal (UIDP/04279/2020).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe author would like to thank Elton José de Souza from Ilha\u0026nbsp;Solteira\u0026nbsp;School\u0026nbsp;of Engineering – UNESP and Marcia Graeff for Bauru School of Dentistry – USP for scanning electron microscopy and confocal microscopy support, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRediT (Contribuidor Roles Taxonomy)\u003c/p\u003e\n\u003cp\u003eConceptualization: [Jesse Augusto Pereira, Aimée Maria Guiotti, Jonas Lobato Duarte, Ana Paula Ramos, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Methodology: [Jesse Augusto Pereira, Rafael Araújo Rios, Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Ana Paula Ramos, Cristiane Duque]; Formal analysis: [Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Cristiane Duque]; Funding acquisition: [Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Project administration: [Ana Paula Ramos, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Supervision: [Aimée Maria Guiotti, Ana Paula Ramos, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Validation: [Ana Paula Ramos, Anil Kishen, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Visualization: [Jesse Augusto Pereira, Rafael Araújo Rios, Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Ana Paula Ramos, Aimée Maria Guiotti, Anil Kishen, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]; Writing–Original Draft: [Jesse Augusto Pereira, Jonas Lobato Duarte, \u0026nbsp; Cristiane Duque]; Writing–Review \u0026amp; Editing: [Jesse Augusto Pereira, Rafael Araújo Rios, Jonas Lobato Duarte,Graciele Ribeiro de Moraes, Ana Paula Ramo, Aimée Maria Guiotti, Anil Kishen, Luís Octavio Regasini, Marlus Chorilli,Cristiane Duque]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHargreaves KM, Geisler T, Henry M, Wang Y (2008) Regeneration potential of the young permanent tooth: what does the future hold? 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FEBS Lett 462:392\u0026ndash;6. https://10.1016/s0014-5793(99)01561-6\u003c/li\u003e\n\u003cli\u003eCaselli A, Cirri P, Santi A,Paoli P (2016) Morin: a promising natural drug. Curr Med Chem 23:774-791. https://10.2174/0929867323666160106150821\u003c/li\u003e\n\u003cli\u003eAkrawi SH, Gorain B, Nair AB, Choudhury H, Pandey M, Shah JN, Venugopala KN (2020) Development and optimization of naringenin-loaded chitosan-coated nanoemulsion for topical therapy in wound healing. Pharmaceutics 12:893. https://10.3390/pharmaceutics12090893.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial activity, oral biofilms, cytotoxicity, morin, nanoemulsions","lastPublishedDoi":"10.21203/rs.3.rs-5923566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5923566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eConsidering the lack of medications with a wide range of therapeutic effects for the endodontic treatment of immature teeth, this study aimed at synthesizing two morin (Mo) derivatives and testing their cytotoxicity and effect on multispecies biofilm in solution and loaded in nanoemulsions (NE).\u003c/p\u003e\u003ch2\u003eMaterial and methods\u003c/h2\u003e \u003cp\u003eMinimum inhibitory and bactericidal concentration (MIC/MBC) of Mo, penta-acetylated Mo (Ac-Mo), Mo complexed with strontium (Sr-Mo) and control chlorhexidine (CHX) were determined against some oral bacteria. NE were physiochemically characterized by analysis of particle size, polydispersity index and zeta potential. NE containing Mo, its derivatives and CHX were evaluated against multispecies biofilms by bacterial counts, scanning electron microscopy and confocal microscopy. The cytotoxicity of the compounds and NE was also determined in fibroblasts using resazurin assays. The data were statistically evaluated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll compounds presented bactericidal action against all bacteria tested (MIC/MBC from 0.125 to 1 mg/mL). Metabolic activity of fibroblasts was higher than 70% after treatment with compounds at 0.25mg/mL or lower. NE showed good droplet size distribution in nanoscale and stability. The highest bacterial reduction in multispecies biofilms was observed in NE\u0026thinsp;+\u0026thinsp;Ac-Mo, followed by NE\u0026thinsp;+\u0026thinsp;Mo, CHX and NE\u0026thinsp;+\u0026thinsp;Sr-Mo groups. All NE diluted at 12.5% did not affect fibroblast metabolism after 24h of treatment.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMorin and its derivatives, either alone or loaded in nanoemulsions, were effective against oral bacteria and showed cytocompatibility at lower concentrations.\u003c/p\u003e\u003ch2\u003eClinical Relevance:\u003c/h2\u003e \u003cp\u003eNanoemulsion containing penta-acetylated morin could be an alternative intracanal medication for reducing residual bacteria between clinical appointments in regenerative endodontic approaches for immature teeth.\u003c/p\u003e","manuscriptTitle":"Clinical Oral Investigations Biological and Physicochemical Characterization of Nanoemulsions Containing Morin and its Derivatives for Endodontic Purposes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 17:59:40","doi":"10.21203/rs.3.rs-5923566/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8e213d7-c573-4b31-8620-02ab64f6e4f9","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T07:39:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-31 17:59:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5923566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5923566","identity":"rs-5923566","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}