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Published in final edited form as: ACS Appl Mater Interfaces. 2024 Jul 15;16(29):37613–37622. doi: 10.1021/acsami.4c06618

Surface-Charge Tuned Polymeric Nanoemulsions for Carvacrol Delivery in Interkingdom Biofilms

Jungmi Park 1,§, Zhenting Xiang 2,3,§, Yuan Liu 2,4, Cheng-Hsuan Li 1, Chider Chen 5, Harini Nagaraj 1, Tiffany Nguyen 1, Ahmed Nabawy 1, Hyun Koo 2,3,6,*, Vincent M Rotello 1,*
PMCID: PMC11624604  NIHMSID: NIHMS2039311  PMID: 39007413

Abstract

Biofilms, intricate microbial communities entrenched in extracellular polymeric substance (EPS) matrices, pose formidable challenges in infectious disease treatment, especially in the context of interkingdom biofilms prevalent in the oral environment. This study investigates the potential of carvacrol-loaded biodegradable nanoemulsions (NEs) with systematically varied surface charges—cationic guanidinium (GMT-NE) and anionic carboxylate (CMT-NE). Zeta potentials of +25 mV (GMT-NE) and −33 mV (CMT-NE) underscore successful nanoemulsion fabrication (~250 nm). Fluorescent labeling and dynamic tracking across three dimensions expose GMT-NE’s superior diffusion into oral biofilms, yielding a robust antimicrobial effect with 99.99% killing for both streptococcal and Candida species and marked reductions in bacterial cell viability compared to CMT-NE (~4-log reduction). Oral mucosa tissue cultures affirm the biocompatibility of both NEs with no morphological or structural changes, showcasing their potential for combating intractable biofilm infections in oral environment. This study advances our understanding of NE surface charges and their interactions within interkingdom biofilms, providing insights crucial for addressing complex infections involving bacteria and fungi in the demanding oral context.

Keywords: dental caries, antimicrobials, nanoemulsions, essential oil, interkingdom biofilm

Graphical Abstract

graphic file with name nihms-2039311-f0008.jpg

INTRODUCTION

Biofilms are communities of microbial cells embedded in self-secreted extracellular polymeric substance (EPS) matrices, causing a variety of infectious diseases.1 The EPS matrix promotes drug resistance by protecting resident microbes against antimicrobial agents and host immune response,2 posing unique therapeutic challenges.3 Polymicrobial interactions within the biofilms, especially between fungi and bacteria, often enhance biomass and virulence, leading to severe infections.4 The intricate nature of these interkingdom communities coupled with concomitantly complex EPS imposes formidable hindrance to eradication compared to monomicrobial counterparts.5,6,7,8 The combination use of antibiotics and antifungals simultaneously or sequentially can have a synergistic effect on biofilms, however, drug penetration and antimicrobial resistance remain a challenge in interkingdom biofilm control.9,10

Candida albicans is a leading pathogenic species known to contribute to polymicrobial infections.11 Significantly, C. albicans has been frequently detected with high levels of the bacterium Streptococcus mutans, forming a hyper-virulent interkingdom biofilm associated with severe early childhood caries (S-ECC).12 Previous studies have revealed that C. albicans and S. mutans develop a symbiotic relationship with emergent properties, including enhanced microbial colonization, a faster growth rate, densely-packed biofilm structure within EPS-rich matrices, and increased acidogenicity.13 This symbiosis consequently confers increased resilience to conventional antimicrobial approaches, rendering the biofilms more resistant to eradication.14,15

Phytochemicals such as carvacrol (oregano oil), are promising therapeutics for bacteria and fungi.16,17,18 These naturally derived bioactives feature broad-spectrum antimicrobial activity,19 high biocompatibility,20 and have a high barrier to resistance development.19, 21 Carvacrol, approved by the FDA for use in food, possesses both antifungal and antibacterial properties, making it effective against the growth and proliferation of both C. albicans and many bacterial species.22 Carvacrol interacts through surface electrostatics and disrupts membrane integrity reducing ergosterol content.23 This dual activity is particularly advantageous in the context of polymicrobial infections involving interkingdom biofilms. However, the current strategies utilizing carvacrol are limited due to their poor solubility24 and hindered permeability into intractable biofilms.25

Recent advances in nanomaterials offer strategies for delivering poorly soluble antimicrobial agents,26,27 with polymeric nanoparticles providing versatile carriers due to their tunable structural and physicochemical properties.28,29,30 Choice of polymer architecture allows for modulation of key features including particle size, solubility, and surface charge.31 Previously, we synthesized a cationic amphiphilic polymer, PONI-GMT (Scheme 1) and used it to fabricate biodegradable nanoemulsions (NEs) for efficient delivery of antimicrobial essential oils against monospecies biofilms.32 However, it is remains unclear if electrostatic interaction plays a role in mediating polymer binding and penetration into negatively charged microbial cell membranes33,34 or biofilms.35

Scheme 1.

Scheme 1.

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Schematic representation of the penetration profiles of a) cationic nanemulsion (GMT-NE) and b) anionic nanoemulsion (CMT-NE) across oral biofilms.

Hence, in this study, we probed how surface charges affect penetration and subsequent therapeutic activities against recalcitrant interkingdom biofilms. We synthesized polymers bearing anionic and cationic functional groups. Then, two distinct charged NEs loaded with carvacrol were fabricated and investigated: 1) positively charged NEs with cationic guanidinium group (GMT-NE) and 2) negatively charged NEs with anionic carboxylate groups (CMT-NE). Importantly, the zeta potential was measured at +25 mV (GMT-NE) and −33 mV (CMT-NE). Both systems manifest consistent hydrodynamic size of ~250 nm, indicating successful fabrication of NEs differing only in discrete surface charge attributes. Fluorescent labeling of the NEs provided insight into their spatial and temporal dynamics in oral biofilms, revealing accelerated diffusion of carvacrol oil with positively charged GMT-NEs compared to the CMT-NE analog. Significantly, this disparity in diffusion translated into a stronger biocidal effect by GMT-NE within interkingdom biofilms, with substantial reductions in cell viability that are unattainable without the NE-mediated delivery. Additionally, the three-dimensional (3D) tissue cultures affirmed the biocompatibility of both NEs validating their potential for safe application in antibiofilm treatments. Taken together, the exploration of surface charges of NEs and their interactions within the interkingdom biofilms augments our insights into strategic elements governing therapeutic efficacy against interkingdom infections as well as bacterial and fungal biofilms.

RESULTS AND DISCUSSION

Synthesis and Characterization of Nanoemulsions GMT-NE and CMT-NE

Nanoemulsions utilizing poly(oxanorbornenimide) polymers (PONIs), that integrate essential oils provide a stable, easily controlled platform for antimicrobial delivery. PONI-GMT is functionalized with positively charged functional groups guanidinium, enabling attractive interactions with bacteria and biofilms through electrostatic interactions. The nanoemulsion system generated by PONI-GMT (PONI-GMT, Mn: 38600, PDI: 1.78) (GMT-NE) shows strong antibiofilm activity in bacterial biofilms supported by efficient delivery of essential oils through electrostatic interactions with negatively charged biofilm components.36 In the current study, we probe the impact of the surface charge of nanoemulsions on the eradication of a bacterial-fungal biofilm relevant to oral biofilms. The anionic polymer PONI-CMT was synthesized as a counterpart to PONI-GMT to generate negatively charged nanoemulsions from the carboxylate groups (10%) with the rest of tetraethylene glycol monomethyl ether and maleimide part (PONI-CMT, Mn: 40900 g/mol, PDI: 1.85, Figure S1–S4). We hypothesize utilizing the carboxylate group in the polymer would generate negatively charged nanoemulsions, as the pKa of carboxylic acid ranges from the 4–7. To generate carboxylate, small portion of potassium carbonate solution was added to PONI-CMT solution to adjust pH to 8. Negatively charged nanoemulsions (CMT-NEs) were fabricated with PONI-CMT and characterized for size and surface charge. The CMT-NE showed a consistent size of ~250 nm, similar to GMT-NE (~220 nm), combined with a narrow size distribution (Figure 1a, PDI < 0.26). TEM further confirmed the size and showed the system has spherical morphology (Figure 1b). Zeta potential measurement showed a negative charge for CMT-NE (−33 mV) and for a positive charge GMT-NE (+25 mV) (Figure 1c and d). The consistent size of the two systems enables probing the effect of the differently charged NEs on biofilm interactions.

Figure 1.

Figure 1.

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Characterization of charged nanoemulsions GMT-NE and CMT-NE by a) Dynamic light scattering histogram in PBS. b) Transmission electron microscopy images of GMT-NE (top) and CMT-NE (bottom) and zeta potential measurement of c) GMT-NE and d) CMT-NE.

Stability of GMT-NE and CMT-NE in human saliva

Efficient delivery of therapeutic payloads at infectious sites can be attributed to stability of system in physiological oral environment. We evaluated the colloidal stability of nanoemulsions in human saliva by monitoring particle size via dynamic light scattering (DLS). Pooled human saliva (Innovative Research IR100044P) was diluted in various concentrations (100% saliva, 50% saliva, 20% and 0% in phosphate buffer saline (PBS)) and nanoemulsions were incubated for 30 minutes at 37 °C. Hydrodynamic sizes of nanoemulsions were measured after incubation. As shown in Figure 2, there was a minimal (~20 nm) increase in size for CMT-NE, possibly due to the presence of saliva proteins.37 A more drastic increase was observed in GMT-NE, with up to a 150 nm increase in diameter, suggesting some level of NE particle Ostwald-ripening-like process.38,39 Given the heterogeneous environment in biofilms, we also assessed the stability of our systems at different pH (Figure S4). Particle sizes were monitored at 3 different pH ranges (pH=6, pH 7, and pH 8). Only at pH 8, the size was increased, with GMT-NE showing a modest increase in diameter. Overall, both NEs showed good stability in saliva and at a wide range of pH values.

Figure 2.

Figure 2.

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Stability and degradability of CMT-NE and GMT-NE. DLS size distribution changes after incubation with human pooled saliva varying concentrations (0%, 20%, 50%, 100% v/v in PBS) of (a) CMT-NE and (b) GMT-NE.

Penetration comparison of GMT-NE and CMT-NE

Biofilms are characterized by the presence of extracellular polymeric substances (EPS), which play a crucial role in providing structural integrity and impeding the penetration of antimicrobial treatments.40 In interkingdom biofilms, the complexity escalates due to the synergistic interactions between bacteria and fungi, which notably enhance biofilm biomass. Within these biofilms, bacterial cluster, yeast cells and hyphal forms are intertwined within a dense matrix of EPS, leading to a robust, highly protected environment that poses a formidable challenge for treatment.15,41

To address this issue, it is imperative to achieve sufficient penetration of antimicrobials into the biofilm architecture to effectively eliminate biofilm-associated infections.42 The EPS of S. mutans may exhibit a negative charged nature, attributed to the incorporation of lipoteichoic acid (LTA) and extracellular DNA (eDNA).43 Additionally, it is important to consider that both bacteria and fungi typically possess a net negative charge on their cell surfaces, primarily due to carboxyl, amino, and phosphate groups, as well as surface glycans.44,45,46 Thus, we hypothesized that the penetration of NEs with different charges would be influenced by electrostatic interactions with charged components present in the biofilm matrix.

To address this hypothesis, we investigated the penetration capabilities of GMT-NE and CMT-NE using high-resolution confocal microscopy across three dimensions. Nile red was encapsulated within the NEs to facilitate dynamic tracking of nanoparticle penetration within the 3D architecture of biofilms. Firstly, we examined the spatiotemporal distribution of NEs within the microcolony from a cross-sectional perspective (Figure 3a). We focused on the middle layer of the microcolonies with a circular-like shape and ‘trisected’ into three regions: center, mid-center and peripheral regions. We profiled fluorescent intensity values distributed across the entire microcolony section after 30 min of nanoemulsion penetration. As shown in Figure 3b, the peripheral region exhibited higher intensity compared to the center, suggesting that the penetration occurred from the outer surface towards the inner area. Interestingly, GMT-NE showed higher intensity in both the center and mid-center regions. In contrast, CMT-NE highly concentrated on the peripheral regions with low values throughout the mid-center indicating limited penetration compared to the positively charged GMT-NE (Figure 3b).

Figure 3.

Figure 3.

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Cross-sectional penetration analysis of nanoemulsion within the microcolony. (a) Three-dimensional schematic diagram for confocal imaging and cross-sectional sections within the microcolony, which were divided into center-, mid-center- and periphery region. (b) Fluorescence-intensity distribution analysis of Nile-red in cross-sectional sections within the microcolony. (c) Time-lapse confocal imaging of nanoemulsion (labeled with Nile-red, in red) penetrating into microcolonies (labeled with SYTO 9, in green) in cross-sectional view.

To further investigate the penetration kinetics, we employed time-lapse imaging as illustrated in Figure 3c. No discernible red fluorescence was observed in either group initially (0 min). After 1 min, fluorescence began to accumulate in the peripheral regions of the microcolony. Over time, the fluorescence of GMT-NE progressively accumulated deeper into the central region. At 30 min, the fluorescence distributed throughout the entire microcolony, indicating the successful penetration of GMT-NE. In contrast, fluorescence from CMT-NE predominantly remained in the peripheral regions, with minimal penetration into the microcolony.

Next, we assessed the orthogonal distribution of the NEs within the microcolony from top to bottom layers (Figure 4a). Similarly, the fluorescence from GMT-NE distributed across the thickness of intact microcolony after 30 min of nanoemulsion penetration, with a significantly higher intensity in the top and middle layers (Figure 4b). In contrast, the majority of fluorescence signal from CMT-NE remained at the top layers of the microcolony, with minimal fluorescence in the middle or bottom layers (Figure 4b). Dynamic fluorescence tracking further revealed that GMT-NE quickly penetrated the microcolony, gradually reaching the bottom layers (Figure 4c). Conversely, CMT-NE predominately accumulated in the upper layers, forming a distinctive arch-like structure enveloping the microcolony (Figure 4c).

Figure 4.

Figure 4.

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Orthogonal penetration analysis of nanoemulsion within the microcolony. (a) Three-dimensional schematic diagram for confocal imaging and orthogonal sections within the microcolony, which were divided into top-, middle- and bottom layer.

(b) Fluorescence-intensity distribution analysis of Nile-red in orthogonal sections within the microcolony. (c) Time-lapse confocal imaging of nanoemulsion (labeled with Nile-red, in red) penetrating into microcolonies (labeled with SYTO 9, in green) in orthogonal view.

The results indicate a superior penetration capability of GMT-NE into biofilms compared to CMT-NE, which aligns with previous findings underscoring the enhanced penetration of positively charged nanoparticles relative to their anionic or neutral counterparts.47 The discrepancy in penetration of these two systems is likely to be attributed to a ‘catch-and-release’ phenomenon within the anionic EPS matrix.48

Efficacy of two different charged NEs against interkingdom biofilms

To evaluate the antibiofilm efficacy of GMT-NE and CMT-NE, we analyzed cell viability within interkingdom biofilms after different treatment durations (10 min, 30 min, and 60 min). We found that GMT-NE significantly reduced microbial cell viability (CFU/ml) in biofilms by 1 log of both S. mutans and C. albicans within 10 min compared to control, whereas CMT-NE modestly reduced the cell viability of S. mutans only (Figure 5a) Three–dimensional schematic diagram for confocal imaging and orthogonal sections within the microcolony, which were divided into top-, middle- and bottom layer. $$PARABREAKHERE$$(b) Fluorescence–intensity distribution analysis of Nile–red in orthogonal sections within the microcolony. (c) Time–lapse confocal imaging of nanoemulsion (labeled with Nile–red, in red) penetrating into microcolonies (labeled with SYTO a). When the treatment time was extended to 30 min, we found that GMT-NE showed more pronounced killing of both microbes than CMT-NE, with GMT-NE approximately 20-fold and 10-fold more effective against S. mutans and C. albicans, respectively (Figure 5b). Remarkably, GMT-NE resulted in complete microbial eradication after 60 min treatment (undetectable viable cells, Figure 5c). However, a substantial population of S. mutans (3-log CFU) persisted in CMT-NE group.

Figure 5.

Figure 5.

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Antibiofilm effect of NEs topical treatment on fungal and bacterial species. Viable cells (CFU) of C. albicans and S. mutans following exposure to NEs for (a) 10 min, (b) 30 min, and (c) 60 min. Data are presented as mean ± standard deviation. The quantitative data were subjected to one-way analysis of variance (ANOVA) with Dunnett post hoc test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, not significantly different; n.d., nondetectable (below detection limit).

To exclude the possibility that the killing effect is caused by the free carvacrol oil or polymer, we tested their antimicrobial effects. The data showed that neither free carvacrol oil nor the polymer killed S. mutans or C. albicans (Figure S5). The results align with previous studies indicating that the antimicrobial efficacy of carvacrol can be significantly enhanced when incorporated into delivery systems.49 Carvacrol inherently has poor solubility and limited permeability into biofilms, and the NE system improves its solubility, penetration and facilitates sustained release, thereby enhancing its ability to kill biofilm cells​​. These results indicate that GMT-NE is more effective against interkingdom biofilms than CMT-NE, presumably due to superior biofilm penetration capabilities and positively charged moieties that enables direct interaction with larger population of microbial cells.39 Conversely, the restricted penetration of CMT-NE hinders its bioavailability within the biofilm while limiting direct contact with the microbes.

Cytotoxicity of GMT-NE and CMT-NE in vitro

The biocompatibility of NEs was assessed via terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, and histological analysis. A three-dimensional (3D) culture of murine oral mucosa tissue was used as an experimental model to recapitulate the biological features of human oral mucosa. As shown in Figure 6a, TUNEL staining (in green) label nuclei indicating cell death due to apoptosis. Neither GMT-NE nor CMT-NE induced cell apoptosis, as evidenced by the low percentages of TUNEL positive cells that were similar to control group (Figure 6a and Figure 6b). Histological analysis of murine oral mucosa tissues showed no morphological and structural changes (Figure 6a, fourth panel). Additionally, a 3D spheroid model was employed to further investigate the biocompatibility. The NEs showed biocompatibility with the multicellular spheroid structure without inducing apoptosis (Figure S6), confirming lack of any cytotoxicity by both NEs.

Figure 6.

Figure 6.

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Cytotoxicity testing for NEs using tissue culture model. (a) TUNEL assay and histological staining in oral mucosal tissue model treated with NEs. Tissue sections were stained with TUNEL assay kit to label fragmented DNA in apoptotic cells (green) and counterstained with DAPI to visualize nuclei (blue). Tissue sections were also stained with H&E to evaluate the histopathology of mucosal tissue following the exposure to NEs. (b) Quantitative analysis of apoptotic nuclei showing the percentage of TUNEL-positive cells relative to total cells (as performed with COMSTAT). Data are presented as mean ± standard deviation of 5 independent replicates. The quantitative data were subjected to one-way analysis of variance (ANOVA) with Dunnett post hoc test. ns, not significantly different.

CONCLUSIONS

In summary, this study highlights carvacrol-loaded antimicrobial nanoemulsions (NEs) against the intricate challenges posed by interkingdom biofilms. By comparing the effect of positive (GMT-NE) and negative surface charged (CMT-NE) carvacrol-loaded NEs, we demonstrate the superior antimicrobial effects of GMT-NE relative to anionic CMT-NE, showing faster and more effective eradication to both bacterial and fungal components simultaneously. Notably, this multifunctional system is stable in saliva and exhibits remarkable penetration capabilities, effectively targeting dense biofilms laden with bacteria, fungi, and extracellular polymeric substances, which cannot be achieved by carvacrol alone. Furthermore, both systems showed no adverse effects against mammalian cells in tissue and 3D culturing models, demonstrating their biocompatibility. The positively charged GMT-NE presents a promising approach to combat the recalcitrance of interkingdom biofilms. This work underscores the significance of tailored nano-based delivery systems in tackling the multifaceted nature of biofilm infections to deliver FDA-approved agents, providing a valuable framework for future advancements in this critical field with high clinical translational potential.

EXPERIMENTAL SECTION

Preparation of the Nanoemulsions GMT-NE and CMT-NE

NEs were prepared through emulsification of a suspension of Nile red in carvacrol into an aqueous polymer solution. For prepared the cationic nanoemulsion, suspension of Nile red in carvacrol (3 μL, 1 mg/ 1000 μL) was added to the PONI-GMT aqueous solution (497 μL, 6.04 μM) then emulsified for 50 seconds using an amalgamator. The anionic nanoemulsions was prepared in a similar way but using PONI-CMT aqueous solution (497 μL, 6 μM). The concentration of these nanosemulsion stock solution were defined as 100 v/v % (39 mM of carvacrol).

Characterization of the Nanoemulsions GMT-NE and CMT-NE

Dynamic light scattering

The hydrodynamic diameter of nanoemulsions was measured in triplicate using DLS (Malvern Zetasizer). Sample was prepared by adding 100 μL of the nanoemulsion to 900 μL of 1x phosphate buffered saline (PBS). Pooled Human Saliva (Innovative Research, MI) was diluted in PBS, and nanoemulsion was diluted in saliva solution (20 %, 50 %, 100 %, v/v) and prepared in different pH adjusted solutions to afford pH 6, 7, and 8.

Zeta potentials

The zeta potential of nanoemulsions was measured in triplicate using DLS (Malvern Zetasizer). Sample was prepared by adding 100 μL of the nanoemulsion to 900 μL of 10 mM NaCl.

Microbial strains and culture conditions

Candida albicans SC5314, a well-characterized fungal strain and Streptococcus mutans UA159 serotype c (ATCC 700610), a virulent cariogenic pathogen, were used for interkingdom biofilm experiments in the study. Both microorganisms were grown to mid-exponential phase in ultrafiltered (10-kDa-molecular-mass-cutoff membrane; Millipore, MA, USA) tryptone-yeast extract broth containing 2.5% tryptone and 1.5% yeast extract (UFTYE; pH 5.5 and pH 7.0 for C. albicans and S. mutans, respectively) containing 1% (wt/vol) glucose at 37 °C, 5% CO2 and harvested prior to use in the biofilm assays.50

In vitro interkingdom biofilm model

Biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (surface area = 2.7±0.2cm2; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) as described previously.51 To mimick the smooth surfaces of the pellicle-coated tooth, the sHA discs were suspended vertically in 24-well plates using a custom-made wire disc holder. Each sHA disc was inoculated with approximately 2×106 CFU of S. mutans per ml and 2×104 CFU of C. albicans per ml in UFTYE broth with 1% (w/v) sucrose and incubated at 37°C under 5% CO2; the proportion of inoculated microorganisms was similar to that found in saliva samples from children with ECC.52 The culture medium was changed twice daily (at 18 and 28h) until the end of the 42 h experimental period.

Biofilm treatment and quantitative analysis

At the end of the experimental period (42 h), the biofilms were topically treated by placing them in 2.8 ml of nanoemulsions (10 mM, 732 μg/mL) in phosphate-buffered saline (PBS; pH 7.4) or vehicle-control (PBS only) for 10 min, 30 min or 60 min. The treated biofilms were dip-washed three times in 0.85 % NaCl to remove excess and unbound agents, and then collected and analyzed for biological assessment. Briefly, the biofilms were removed and homogenized via a combination of water bath sonication and probe sonication (30s pulse at an output of 7W; Branson Sonifier 150, Branson Ultrasonics, Danbury, CT, USA), providing optimum dispersal and maximum recoverable counts without killing microbial cells.53 The homogenized suspension was subjected to determine the total number of viable cells in each treated biofilm by counting the colony forming units (CFU) on blood agar plates after serial dilution of the homogenized suspension.

Biofilm penetration and quantitative analysis

Biofilms were formed on sHA discs inoculated with approximately 2×105 CFU of S. mutans per ml in UFTYE broth containing 1% (w/v) sucrose and cultured for 19 h as detailed above. Bacterial cells were stained with 2.5μM SYTO 9 green fluorescent nucleic acid stain (485/498nm; Molecular Probes Inc., Eugene, OR, USA). The NEs labeled via Nile red were added to the biofilm at a concentration of 10 mM (732 μg/mL) to visualize the localization of the NEs within the 3D architecture of the microcolony.54 An upright single-photon laser scanning confocal microscope (LSM800; Zeiss, Jena, Germany) was used and images were acquired in the same field of view at 0, 1, 5, 10, 20, and 30min with a 20× (numerical aperture, 1.0) water immersion objective. Each component was illuminated sequentially to minimize cross talk as follows: SYTO 9 was excited at 488nm and collected by a 480/40-nm emission filter, and Nile red was excited at 561nm and collected by a 575/40-nm emission filter. The confocal images were acquired from at least 3 randomly selected spots of each sample at each time point. ImageJ software (version 1.48) was used to create 3D renderings of biofilm architecture and quantitative analysis.

Biocompatibility assessments

To test the biocompatibility of nanoemulsions in vitro, two different types of tissue three-dimensional (3D) cultures were used. We first performed a whole-organ explant culture of murine oral mucosa as detailed previously.55 Briefly, mucosal tissue (4 mm × 2 mm) was obtained from the palate of C57BL/6 mice and cultured in MEM α media (15% FBS, 2 mM L-glutamine, 100 μM ascorbic acid, 100 U/mL penicillin, and 100 μg/mL streptomycin) at 37 °C with 5% CO2 for 24 h. The second model we used for biocompatibility assessment is 3D spheroids. Briefly, human gingival tissue-derived mesenchymal stem cells (GMSCs) were isolated from gingival tissues obtained during third molar extractions at the University of Pennsylvania following the approved Institutional Review Board (IRB) protocol (IRB#816238).56 GMSCs were expanded up to passage five in Minimum Essential Medium Alpha (MEM α) media (containing 15% FBS, 2 mM L-glutamine, 100 μM ascorbic acid, 100 U /mL penicillin, and 100 μg/mL streptomycin) before using in the experiments.57 GMSCs (106/mL) were seeded in ultra-low attachment tubes and grew for 3 days in a humidified tissue culture incubator (37 °C, 5% CO2) to form 3D spheroids.

The 3D spheroids were harvested and topically treated by placing them in 2.8 ml of nanoemulsions in phosphate-buffered saline (PBS; pH 7.4) or vehicle-control (PBS only) for 30 min. The samples were collected after treatment and fixed in 4% paraformaldehyde (PFA), followed by embedding in OCT compound (Sakura Finetek, Torrance, CA, USA). Frozen sections were sliced and stained with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to detect the dead cells due to apoptosis. For histological analysis, slides were stained with H&E staining.

Statistical analysis

All experiments were performed in triplicate. All data were presented as means ± s.d. and analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. Statistical significance was determined at P < 0.05. GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA) was used to perform and analyze statistical tests.

Supplementary Material

ESI

ACKNOWLEDGEMENTS

This work was supported by the National Institute of Health under R01 AI134770 (V. R.) and R01 DE025220 (H.K.). Y.L and Z.X. are the recipient of the Center for Innovation & Precision Dentistry (CiPD)-Colgate Fellowship funded by Colgate-Palmolive Company.

Footnotes

Supporting Information

• Materials and methods, instrumentation, monomer an polymer characterization, DLS at different pH, effects of free carvacrol and polymer controls on interkingdom biofilm and three-dimensional spheroid culture cytotoxicity test

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