Dental plaque accumulation and inadequate personal oral hygiene are the severe challenges for maintaining oral health in orthodontic patients 26. Reduction of bacterial colonization and removal of dental biofilms as one of the most important etiologic parameters for oral pathological conditions are performed by various techniques, but with limited success 27,28.
Recently, aPDT has been introduced as an alternative therapy for preventing and treating dental caries via control the formation of the microbial biofilm and controlling the incidence of microbial pathogens without the development of resistance 29–31. Several variables such as the type and concentration of photosensitizers, light sources, irradiation parameters, and physiologic condition of the microorganisms studied must be considered for the effectiveness of aPDT 32.
Many in vitro and in vivo studies present high efficiency of aPDT against microorganisms involved in dental caries 33–38. The results of Alqerban 39 study showed that no only aPDT with Riboflavin and Rose Bengal can be used for bonding orthodontic brackets to the tooth surface but also revealed the substantial antibacterial properties against S. mutans. Hugo Panhóca et al. 40 reported aPDT with Curcumin reduces the number of living cells of S. mutans in a biofilm model created on the surface of metallic orthodontic accessories.
The results of previous studies showed that ZnONPs are the most promising next-generation photosensitizer for PDT due to their specific phototoxic effect on tumor areas 41–44. According to Pourhajibgher et al. photo-activated Curcumin doped ZnONPs can use as an orthodontic adhesive additive to control the cariogenic multispecies biofilm, and also reduce their metabolic activity 7.
The biocidal cations of ZnONPs have been hosted on the surfaces and in the cavities of Zeo via ion exchange and can become an efficient nano-photosensitizer in aPDT process. On the other hand, Zeo as stable carriers with high chemical inertness and null toxicity could be increasing the efficacy of photosensitizer via protecting it from oxidation, increasing the permeability of the photosensitizer through the cell membrane, increasing the concentration of photosensitizer molecules in the target cells, and increasing the lifetime of photosensitizer release through a sustained process 45–48.
The possible risks of nano-photosensitizer to human health have raised concerns. These concerns underline the need and importance of assessing their cytotoxicity. In a work reported by Wang et al. 49 the cytotoxic effects of ZnONPs at concentrations of 10, 15, 30, and 100 µg/mL was investigated on different cell types such as human keratinocyte cells (HaCaT), human gingival fibroblast (HGF-1), and human gingival squamous carcinoma cell line (Ca9-22). Their results showed that ZnONPs were less toxic to normal HaCaT and HGF-1 cells, but showed severe toxicity to Ca9-22 cells at concentrations more than 30 µg/mL. Seker et al. 50 and Vergara-Llanos et al. 51 reported ZnONPs exhibited cytotoxic effect at doses of 50–100 µg/mL. In this study, the cell-killing effect of Zeo\ZnONPs as the model photosensitizer at the different concentrations was tested on HuGu cells. Zeo\ZnONPs at 0.5×10− 4 g/L concentration had very low cytotoxicity; 3.8% and 2.4% of HuGu cells are viable following MTT- and SRB-based cytotoxicity assay, respectively. Also, after a 24 h incubation period, 19.4% and 18.7% of HuGu cells were viable at the highest concentration of Zeo\ZnONPs (2×10− 4 g/L) following MTT and SRB assay, respectively. As the results displayed the cytotoxicity of Zeo\ZnONPs was dependent on concentration.
In this study, the hemolytic effect of Zeo\ZnONPs on erythrocytes was assessed and its biocompatibility at the different concentrations was revealed. Babu et al. 52 explored the hemolytic effect of ZnONPs was increased with increasing concentration from 25 to 800 µg/mL in a time-dependent manner. Besides, the data from Ulyanova et al. 53 showed that with a decrease in the concentration of nano-zeo to 1 mg/mL, there is a tendency for the hemolytic activity of the samples to decrease. Therefore, the results obtained from this study are consistent with the results of previous studies.
There are no studies in the literature on using Zeo\ZnONPs- mediated aPDT for the inhibition of polymicrobial biofilms growth in patients undergoing orthodontic treatment. Herein, a significant decrease on the viability of microorganisms was observed when biofilms were exposed to aPDT. The present study showed an interesting result with 2×10− 4 g/L of Zeo\ZnONPs- mediated aPDT, degradation 92% and 78.6% of biofilms, and metabolic activity of polymicrobial biofilms, respectively. In addition, no remarkable difference in the number of log10 CFU/mL was observed between Zeo\ZnONPs- mediated aPDT (2×10− 4 g/L) and CHX.
Following light irradiation, the photosensitizer in its ground singlet state excited and reacts with oxygen to produce ROS. These ROS can be classified into those produced by Type I photochemical mechanism: free radicals such as superoxide radical anion (O2•−) and hydroxyl radicals (HO•) and those produced by Type II photochemical mechanism: singlet oxygen (1O2). They all have damage effects on biomolecules and kill the target cells, resulting in membrane destructive process, metabolic hydroxylation, oxidative DNA damage, carcinogenesis, etc 54. As Zhang et al. 55 reported, the production of ROS is dependent on photosensitizer concentration, as well as, irradiation dose of the light source. In this ex vivo study, the results demonstrate that 2×10− 4 g/L Zeo\ZnONPs can generate ROS within polymicrobial biofilms after blue laser irradiation about 1.87-fold.
To the best of our knowledge, this is the first time to evaluate the remineralization potential of enamel carious lesions. Enamel samples treated with Zeo\ZnONPs-based aPDT exhibited enhanced surface microhardness when compared with samples treated with artificial saliva alone. In this study, like several previous studies, the NaF varnish was effective for enamel remineralization 56–58. However, aPDT using 2×10− 4 g/L Zeo\ZnONPs and the NaF varnish groups were not statistically different (P > 0.05).