Characterization of MCDs and TCDs
In this study, we synthesized TCDs and MCDs based on anti-anaerobic drugs Tinidazole and Metronidazole, respectively, through a simple green hydrothermal synthetic method [20, 56]. The TEM images and the size distribution by Dynamic Light Scattering (DLS) of TCDs in (Fig. 2 a1 ,b1) and MCDs in (Fig. 2 a2 ,b2) illustrated that the size of TCDs is almost uniform with 16.5 nm and 15.1 nm as the predominant diameters for TCDs and MCDs, respectively. Furthermore, the nanoparticles retained the uniform hydrodynamic size in water. Fig. 2 c1 show the UV-Vis spectra of TCDs and Tinidazole, and Fig. 2 c2 showed the UV-Vis spectra of MCDs and Metronidazole. The pro-drug Tinidazole exhibited two broad absorption peaks at 227 nm and 315 nm, ascribed to π–π*(C=N) and n–π*(C–N-C), respectively. While the prepared TCDs showed only one peak at 287 nm, which could be credited to n–π* (C–O). The result of MCDs and Metronidazole in Fig. 1 c2 is consistent with previous research reports .
Then the elemental composition and main functional groups of TCDs and MCDs were identified, respectively. The results of EDS spectra of Tinidazole and TCDs (Fig. 3a1, b1) indicated that the TCDs was mainly consisted of carbon (61.0 atomic percentage (at) %), nitrogen (14.2 at%) oxygen (20.7 at%) and sulfur (4.1 at%). However, the Tinidazole mainly comprised of carbon (54.1 at%) and nitrogen (20.4 at%), which demonstrated that the content of carbon rose after the reaction and the carbon polymerization of TCDs, indicating the successful synthesis of carbon dots. In addition, as shown in Fig. 3 a2, b2, the element composition of the metronidazole included (C (48.76 at%), O (26.43 at%), N (24.81 at%)) before reaction, but the MCDs was mainly consisted of C (59.43 at%), O (22.35 at%), N (18.22 at%) after preparation, which was consistent with the results of TCDs. Then the photoluminescence (PL) properties of TCDs and MCDs were measured with the excitation wavelengths from 380 nm to 480 nm. As shown in Fig. 3 c1, the TCDs have an excellent emission intensity at the excitation of 400 nm and the emission wavelength of 492 nm. And the insert image in Fig. 3 c1 presenting the TCDs showed blue-green fluorescence with 492 nm. In Fig. 3 c2, the optimal emission of TCDs was at 463 nm under 400 nm excitation. The FTIR spectra (Fig. 3 d1) of Tinidazole exhibited the absorption bonds at 3420, 3161, 1611–1511, 1369,1131–1264 cm−1, which was attributed to the stretching vibrations of v(N–H), v(=C–H), v(–NO2) and v(C–N). Furthermore, some original functional groups were still retained under high temperature and high pressure through the analysis of the FTIR spectrum of the TCDs (Fig. 3 d1), such as stretching vibrations of N-H at around 3417 cm−1 with strong and broad absorption peaks, which may be caused by the bending vibrations of N–H, the bonds at 1540–1470 cm−1 and 1010–1090 cm−1 corresponded to C–N and C–H, respectively. The results of metronidazole and MCDs in Fig. 3 d2 resemble previous findings . The formation of these chemical bonds was mainly due to the physical change and chemical reaction processes. Notably, -NO2 was also detected in TCDs, but its relative content in nanoparticles was lower than that in Tinidazole, perhaps some of them were reduced. It has been reported that anaerobic bacteria can reduce the nitroimidazole nitro group to hydroxylamine by electron transfer protein, which further leads to reaction with bacterial proteins and DNA to prevent the synthesis of all nucleic acids [9, 10, 40]. These results demonstrated that TCDs were successfully prepared through a simple and green hydrothermal method and still retain the possibility of selective bacteriostatic ability .
Biocompatibility of TCDs and MCDs
Obviously, good biocompatibility and low toxicity of nanomaterials are excellent indicators for biomedical applications. For this purpose, normal human liver L0-2 cells were treated with different concentration of MCDs and TCDs for biocompatibility assay. Fig. 4 showed that the viability of L0-2 cells was more than 80% after 24 h of incubation with TCDs, even when the concentration reached 100 μg mL−1. But the survival of L0-2 cells treated with MCDs below 80% at the same concentrations MCDs. These results demonstrated that TCDs exhibited good biocompatibility and low toxicity.
Selective inhibition of bacterial growth by TCDs and MCDs
As a commonly used antibacterial drug, Metronidazole and Tinidazole are specially effective for gram-negative and anaerobic bacteria [9, 57]. Therefore, we tested the specific inhibitory effect of TCDs and MCDs on proliferation of different bacteria. E. coli, S. aureus, P. gingivalis and P. nigrescens were selected for test the inhibitory zone with different concentrations of TCDs or MCDs. As shown in (Fig. 5 a1-2, b1-2) there were no obvious inhibitory zones formed around the filter paper treated with TCDs or MCDs on the (E. coli and S. aureus) bacterial plats. However, obvious inhibitory zone for P. gingivalis were observed around filter papers treated with TCDs and MCDs, respectively (Fig. 5 d1, d2). It should be note that the MCDs had a significant inhibition on P. nigrescens (Fig. 5 c2), on the contrary, the TCDs in (Fig. 5 c1) had a weaker effect than MCDs on P. nigrescens. To further determine the effect of TCDs and MCDs on the inhibition of the bacteria, different concentrations of TCDs and MCDs were added into liquid media by examining the OD value to monitor the growth of the bacteria. In Fig. 5 e-h, the results showed that the TCDs and MCDs had no significant inhibitory effect on E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) growth, however the TCDs and MCDs both showed excellent antibacterial effects against P. gingivalis growth, consistent with the results of the above inhibition zone experiments. Simultaneously, the MCDs (Fig. 5 g) still had bactericidal activity against P. nigrescens, while the TCDs only showed weaker inhibition on P. nigrescens. These results confirmed that TCDs had extraordinary bactericidal activity against P. gingivalis and showed no significant inhibition on other gram-negative anaerobic bacteria. As expected, TCDs exhibited better specificity inhibition than MCDs for P. gingivalis growth, and the antibacterial effects of TCDs which depended to large extent on the antibacterial activity of the nitro group .
The antimicrobial susceptibility of P. gingivalis to TCDs and MCDs were evaluated by measuring the MIC values. The result of TCDs (Fig. 6 a) demonstrated that the TCDs have remarkable antibacterial ability in P. gingivalis and provided the minimum use concentration for further research. We observed that the growth of P. gingivalis at low concentrations slower than the control group, even when the concentration reached 25 μg/mL, the bacterial growth was nearly completely inhibited and then the growth was declined as the concentration was further increased. While in Fig. 6 b when the concentration of MCDs reached 50 μg/mL, the growth of P. gingivalis treated with MCDs became stationary, and no significant bactericidal activity were emerged until using higher concentrations. However, higher drug use concentrations could lead to bacterial resistance and biocompatibility declined, which ultimately resulted in the limited of applications . Therefore, the TCDs were selected for further research, and these results laid the foundation to further explore the anti-biofilm activity and mechanism of TCDs.
The inhibition effect of TCDs on P. gingivalis biofilm formation
Biofilms could wrapped microorganisms and protected them from the killing of drugs, the protective function were afforded by the extracellular matrix (ECM) and several types of biomolecules (polysaccharides, DNA, and peptides). Because of the protective function of biofilms, the P. gingivalis behave durable resistance and viability, which increased the difficulty of treating oral related diseases [2, 3]. Semi-quantitative analysis of crystal violet is a method for judging the amount of biofilm produced by bacteria through decolorization detection after staining their biofilm by using crystal violet . The biofilm formation experiment results (Fig. 7 a) illustrated that when the number of P. gingivalis at 5×106 CFU/mL, the biofilm biomass was detected in higher formation which compared with the control group, however, the amount of biofilm formation did not increase as the amount of bacteria increased in 7.5×106 even 1×107 CFU/mL. We speculated that the growth of biofilm was reduced because of the limited growth space for P. gingivalis. Simultaneously, it provided a reliable reference value for the quantity of bacterial mass to formed a complete biofilm. The images of P. gingivalis biofilms stained with crystal violet after TCDs treatment shown in Fig. 7 b and Fig. 7 c. the first well completely maintained the production of biofilms without the TCDs treatment, and then the production of biofilms gradually decreased with the increased concentrations of TCDs, and almost completely inhibited the formation of biofilm at the concentration of 150 μg/mL, which was consistent with previous reports . Although Tinidazole also can inhibited the formation of biofilm, but the effect was not as obviously as TCDs (Fig. 7 b, 7 c).
In order to more intuitively evaluate the effect of TCDs on P. gingivalis biofilm formation, we employed confocal laser scanning microscope (CLSM) to analyze the clearance and permeability of different concentrations of TCDs on FITC-labeled P. gingivalis and formation biofilms in 3D and orthogonal fields . As shown in the Fig. 8 a, a dense biofilm was observed in the 3D field of untreated group, and the corresponding orthogonal field of view also showed a higher biofilm thickness. On the contrary, the incomplete biofilms was observed on the surface of confocal dish treated with TCDs of 25 μg/mL, and with the concentration increased, the number of biofilms on the bacteria attached to the surface of dish decreased significantly, and the result of relative quantification for biofilm biomass from fluorescence intensity showed that the biofilms reduced by 75% when the concentration of TCDs reached 200 μg/ml (Fig. 8b). In addition, the quantitative results for average biofilm thickness showed that the relative thickness nearly decreased to 5.4 μm (Fig. 8 c). Therefore, TCDs showed an excellent effect in inhibiting biofilm formation.
Penetration of TCDs to biofilm and inhibit P. gingivalis growth
The target bacteria can escape from the large-scale antibiotics killing owing to common drug particles unable to across the grumous biofilm . As far as we know, there is no biofilm penetration sterilization study have been reported before. Firstly, we constructed a biofilm model for biofilm penetration sterilization experiments in vitro (Fig. 9 a). The 0.22 μm filter membrane were paste on the bottom of the upper-chamber used as basement on which the P. gingivalis were cultured, and then added 1×107 CFU of bacteria and cultured for 48 h to form biofilm on the chamber. When the biofilm model successfully formed, different concentration of TCDs were added into the upper-chamber, as shown in Fig. 9 b, the growth of P. gingivalis cultured in the down-chamber were significant inhibited, suggesting that TCDs could penetrate the biofilm and inhibit the growth of P. gingivalis cultured in the down-chamber. It should be noted that when the concentration of TCDs is below 50 μg/mL, there was no obvious inhibition effect on the growth of P. gingivalis. Interestingly, the concentration above 50 μg/mL of the TCDs showed significantly antibacterial activity on P. gingivalis compared with the control. while Tinidazole does not kill P. gingivalis cultured in the down-chamber. Therefore, we speculated that the TCDs could across the biofilm and caused lethality to bacteria at higher concentration. Furthermore, as shown in Fig. 9 c1-e1 were SEM images of P. gingivalis biofilm treated with different concentrations of TCDs or Tinidazole. The result demonstrated that a dense biofilm with many long strips of fibers on the surface was formed after 72 h incubated with P. gingivalis. After treated with different concentration of TCDs, small pores produced, which facilitating the penetration of TCDs to the biofilm. When the concentration of TCDs reached to 100 μg/ml, the biofilm was destroyed severely (Fig. 9 c1-e1). While Tinidazole does not displayed drastically effect on the biofilm. Therefore, the nanostructures of TCDs may play an important role in penetrating the biofilms by affecting P. gingivalis producing Fibrin, the important components of mature biofilms .
The effect of TCDs on the adhesion of P. gingivalis
The adhesion of the P. gingivalis on dental surfaces was tested in the presence of different concentrations of TCDs and Tinidazole. As showed in Fig. 10 a and 10 b, the adhesion of P. gingivalis was influenced in all the experiments that treated with TCDs from the 12.5 to 200 µg/mL, mean time the stained adherent bacteria in the Tinidazole treatment group did not observed obviously differences compared with the TCDs treatment group. The results revealed that the adherence of P. gingivalis had highly sensitivity to the TCDs, while untreated group had no inhibitory effect on adherence activity of P. gingivalis, similarly, the Tinidazole treatment group hardly to affect the adhesion.
Studies have shown that changes in bacterial surface hydrophilicity can cause alterations in bacterial adhesion. Subsquently, we investigated the effect of TCDs on the hydrophilicity of P. gingivalis. As shown in Fig. 10 d, when the concentration of TCDs increased, the concentration of P. gingivalis in the oil phase was reduced, indicating that the surface hydrophobicity of the bacteria was continuously decreasing, similarly, the absorbance of P. gingivalis in water treated with TCDs significant higher than that of in oil, which was in stark contrast to the pro-drug Tinidazole (Fig. 10 c), suggesting that hydrophilic TCDs can increase the hydrophilicity of P. gingivalis and thus reduce the adhesion of bacteria on biofilms, which consistent with previous reports [53, 59].
Effect of TCDs on genes related with biofilm formation of P. gingivalis
P. gingivalis biofilm formation was regulated by multiple genes. Among them, the long fimbriae (FimA) encoded by FimA play a key role in the adhesion of P. gingivalis, and also affect the formation of biofilm [4, 5]. In addition, arg-gingipain (RgpA, RgpB) and lysine-specific cysteine proteases (Kgp), which are also essential for the processing and maturation of FimA proteins, thus promoting adhesion of P. gingivalis to host tissues. The FimA, Rgp (A, B) and KGP genes were decisive for the pathogenicity of P. gingivalis and also affect the important component of biofilms –Lipopolysaccharide (LPS) [60, 61]. Therefore, we detect the mRNA expression of related genes in P. gingivalis treated with different concentration of TCDs. As shown in Fig. 11, the semi-quantitative analysis results showed that the mRNA expression of FimA RgpA, RgpB and KGP decreased drastically. Therefore, we speculate that the protein expression of FimA, Rgp (A, B) and KGP will be decreased correspondingly, thus affecting the self-assembly of Proteins and preventing formation of biofilms .