Nanoceria Nanoparticles as Synthetic Agglutinins for Dental Plaque Reduction

Streptococcus mutans plays a central role in the development of dental caries by forming biofilms adhered to teeth. A nanoceria aggregate formulation, consisting of chondroitin sulfate A (CeO2-NP-CSA), developed in our laboratory lead by Prof. Russell Pesavento in the College of Dentistry at the University of Illinois Chicago, effectively agglutinated S. mutans cells, thereby restricting in vitro biofilm formation. The recently published study details the synthetic preparation, chemical characterization, biological activity, initial mechanistic insights, and cellular toxicity of CeO2-NP-CSA.

Keywords : Streptococcus mutans, Biofilms, Cerium Oxide, Nanoparticles, Dental Caries

Graphical Abstract Adapted from (Ellepola et al., 2023)

Content

The mouth harbors a multitude of germs, such as bacteria, fungi and viruses which are largely benign. Typically, the body’s immune system and diligent oral hygiene practices such as daily brushing and flossing effectively manage the overgrowth of these microbial populations. Neglecting oral hygiene, however, can allow the pathogenic microorganisms to proliferate disrupting the oral microbiota balance, termed as “microbial dysbiosis,” allowing pathogens to evade the host immune response resulting in infections like tooth decay and gum disease.

Under dysbiotic conditions, Streptococcus mutans is among the major bacteria associated with sites on the tooth surface with dental cavities. Streptococcus mutans’ pathogenicity is attributed to its ability to form tooth-adhered biofilms (i.e., plaque) and ferment dietary sucrose into acids that can demineralize the tooth enamel causing cavities (Lemos et al., 2019). In addition, they produce sticky polysaccharides that aids the attachment of bacteria on to the tooth surface (Bowen and Koo, 2011).

Tooth pastes, oral rinses or varnishes used to treat dental caries contain inorganic agents such as Sodium Floride, Silver Diamine Floride, Stannous Floride or their derivatives. However, these agents with their broad spectrum activity can harm certain healthy oral bacteria as well, and cause significant staining issues of the teeth (Frese et al., 2019). When the treatment population are children with cavities, there is further parental concern over staining (Crystal et al., 2017) and potential concern over toxicity as a result of overdose.

Ongoing research in dentistry and microbiology is actively pushing the boundaries to create novel anti-caries agents. These agents aim to be not strongly “cidal” yet effective, aesthetically pleasing, and composed of affordable materials. They’re formulated to remain stable across various conditions (in storage and in the oral cavity) while actively combating S. mutans and other acidogenic species, dispersing their biofilms and preventing plaque buildup.

Extensive research has been dedicated to exploring the efficacy of metal/metal oxide nanoparticles in combating oral biofilms, owing to their significant surface area and heightened chemical reactivity. In our recent research paper published in ACS Biomaterial Science & Engineering (Ellepola et al., 2023), the synthesis of nanoceria aggregate formulation, consisting of chondroitin sulfate A (CeO2-NP-CSA) is outlined. The initial cerium oxide nanoparticles (i.e., Nanoceria, CeO2-NP [3–5 nm]) are derived from the hydrolysis of 1 N H2[Ce(NO3)6]. The initial strategy was to react the above nanoceria with anionic polymers to afford coated structures that are stabilized by a net negative surface charge. One of the polymers screened herein for the stabilization of nanoceria was chondroitin sulfate A (i.e., chondroitin-4-sulfate, CSA) due to its low cost, biocompatibility, and anionic charge over a wide pH range. We could find no prior reports of CSA-associated nanoceria aggregates.

Initially, we assessed the stability of the CSA coated CeO2-NP (CeO2-NP-CSA) in varying pH conditions and in the presence of NaF, given its usage as the active ingredient in numerous over-the-counter (OTC) and prescription oral healthcare products. We identified that the CSA coating allowed the stability of the CeO2-NP under a wide pH range and in the presence of NaF, attributed to the overall negative charge of the nanoparticle formulation.

In assessing the efficacy of CeO2-NP-CSA in inhibiting biofilm (i.e., dental plaque) formation by S. mutans, we employed a 96-well plate method followed by crystal violet staining (Wen and Burne, 2002). At concentrations as low as 125 µM, CeO2-NP-CSA significantly impeded biofilm formation by S. mutans under sucrose-rich conditions, and in a dose-dependent manner. We assessed the growth of S. mutans in the presence of CeO2-NP-CSA by measuring the optical density, aiming to discern whether the biofilm inhibition stemmed from bactericidal activity. Surprisingly, CeO2-NP-CSA did not hinder bacterial growth, suggesting a non-bactericidal mechanism of action for the nanoparticles.

To probe the potential mechanism underlying CeO2-NP-CSA’s biofilm inhibition, we examined its capacity to induce cell aggregation using microscopy. We noted that CeO2-NP-CSA prompted bacterial cells to rapidly form sizable clumps, thereby hindering their attachment to surfaces and impeding the formation of adherent biofilms.

To assess its toxicity against cell lines pertinent to the human oral cavity, CeO2-NP-CSA underwent testing. In line with clinical relevance, NaF served as the toxicity standard for comparison. Treating human keratinocytes and fibroblasts with equimolar concentrations of CeO2-NP-CSA did not result in a notable increase in toxicity compared to NaF.

In summary, CeO2-NP-CSA exists as an anionic aggregate structure with improved storage properties at a wide pH range with no toxicity towards human cell lines compared to equimolar NaF. CeO2-NP-CSA reduces in vitro biofilm formation of S. mutans under dose-dependent conditions via the induction of nonadherent cell clusters, reducing the initial attachment phase of biofilm formation. Studies are underway that investigate the interaction of CeO2-NP-CSA with the S. mutans cell surface (and associated biomacromolecules) that lead to rapid cell clustering, and efficacy and toxicity studies using rodent models. Our research aims to persist in formulating this nanoparticle into a mouth rinse with the potential to mitigate plaque formation.

References

Bowen, W.H., and Koo, H. (2011). Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 45(1), 69-86. doi: 10.1159/000324598.
Crystal, Y.O., Janal, M.N., Hamilton, D.S., and Niederman, R. (2017). Parental perceptions and acceptance of silver diamine fluoride staining. J Am Dent Assoc 148(7), 510-518.e514. doi: 10.1016/j.adaj.2017.03.013.
Ellepola, K., Bhatt, L., Chen, L., Han, C., Jahanbazi, F., Klie, R.F., et al. (2023). Nanoceria Aggregate Formulation Promotes Buffer Stability, Cell Clustering, and Reduction of Adherent Biofilm in Streptococcus mutans. ACS Biomaterials Science & Engineering. doi: 10.1021/acsbiomaterials.3c00174.
Frese, C., Wohlrab, T., Sheng, L., Kieser, M., Krisam, J., and Wolff, D. (2019). Clinical effect of stannous fluoride and amine fluoride containing oral hygiene products: A 4-year randomized controlled pilot study. Scientific Reports 9(1), 7681. doi: 10.1038/s41598-019-44164-9.
Lemos, J.A., Palmer, S.R., Zeng, L., Wen, Z.T., Kajfasz, J.K., Freires, I.A., et al. (2019). The Biology of Streptococcus mutans. Microbiol Spectr 7(1). doi: 10.1128/microbiolspec.GPP3-0051-2018.
Wen, Z.T., and Burne, R.A. (2002). Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Appl Environ Microbiol 68(3), 1196-1203. doi: 10.1128/aem.68.3.1196-1203.2002.

Kassapa Ellepola, PhD,
Visiting Research Assistant Professor,
College of Dentistry,
University of Illinois Chicago, USA