Multiclawed SiO2 Nano-Antibacterial Agent Based on Charge Inversed Ce6 Ionic Liquid Polymers for Combating Oral Biofilm Infection

Photodynamic antimicrobial chemotherapy (PACT) is a promising therapy against biofilm infection. However, due to the saliva clearance and obstacle of biofilm, the photosensitizer is difficult to concentrate in the infection site; then, the PACT is less effective on oral biofilm infection. In this article, we report a special nano-antibacterial agent (SiO2-PCe6-IL) to solve the bottleneck problem of PACT in treatment of oral biofilm infections. The SiO2-PCe6-IL was composed of SiO2 and poly ionic liquid photosensitizer (PCe6-IL) and had tri-fold features of eliminate biofilm infection: high binding ability, breaking biofilm barriers, and enrichment photosensitizer in infection site. In oral biofilm, the SiO2-PCe6-IL changed to SiO2-PIL + like claws of octopus that could hold tightly with biofilm. Then, the poly-dodecyl on the SiO2-PIL + broke down the barrier of biofilm. The results of HR-MS and zeta potential indicated that SiO2-PCe6-IL could change to positive (SiO2-PIL ) in acidic environment. The interaction forces and morphology results proved that the SiO2-PIL + had a higher affinity to biofilm and could destroy the biofilm structure. Then, the photosensitizer was enriched in biofilm at sites of infection. The in vitro and in vivo experiments showed that SiO2PCe6-IL could effectively eradicate oral biofilm infections and control of dental caries.


Introduction
Oral biofilm infection is most important pathogenic factor of dental caries [1,2]. Thus, eliminating biofilm on the tooth surface is a primary strategy to prevent and treat dental caries [3]. However, with the protection, bacteria in the biofilm is difficult to eradicate by common strategy of mechanical cleaning combined antibiotics. In this background, it is urgently to develop new treatment against oral biofilm infection. One promising therapy is PACT [4]. Compared with traditional treatment, PACT could efficiently control of bacteria and bacterial biofilm infection. However, owing to rapid clearance by saliva and barrier of biofilm, the photosensitizers are not concentrated in the infection site, and the efficacy of PACT against oral biofilm infections is limited [5]. Faced with the dilemma, the concentration of photosensitizer in the site of oral biofilm is the key to improve the PACT efficiency.
An understanding of oral biofilms matrix could offer opportunities to exploit photosensitizer delivery systems [6]. Because oral biofilm develop when microbes accumulate and form structured communities encapsulated within an extracellular matrix such as exopolysaccharides (EPS) [7,8], the sugars are fermented by bacteria within the EPS matrix, then created highly acidic microenvironments and negative charge. The pH values often reach pH~4.5 or even lower in oral biofilm and particularly after exposure to sucrose, starch, and other cariogenic food products [9]. So, photosensitizer delivery system that could response to lower pH microenvironments is an effective method to improve delivery efficiency. Furthermore, many researches on cationic polymers [10], especially on the charge-reversal polymers, indicated that positively charged polymers could combine with extracellular polymeric substances (EPS) [11] and increase concentration of photosensitizer in biofilm. However, due to the dense and thick structure of biofilm, the photosensitizers difficultly penetrated into biofilm; then, photochemical reaction initiated by photosensitizer occurs mainly in the outermost layers of biofilm. The PACT efficiency was not significantly improved.
To overcome these challenges, polyionic liquid that composed of polycation and anion is introduced in previous studies [12,13]. Inspired by octopus claws, the polyionic liquid photosensitizer nanoparticle (SiO 2 -P Ce6-IL ) like arms of octopus was prepared in this work. In acid microenvironment of oral biofilm, the SiO 2 -P Ce6-IL could change to nanoclaws SiO 2 -P IL + and firmly combine to negatively charged EPS to overcome the saliva cleaning. Then, the dodecyl on the SiO 2 -P IL + could pierce through the biofilm and break down the barrier of biofilm. Finally, the photosensitizer was highly concentrated in oral biofilm, and the biofilm infection was eliminated. The high binding energy between positively charged SiO 2 -P Ce6-IL and oral biofilm, excellent breaking ability of polydodecyl, and effective enrichment of photosensitizer were the key factors to effectively eliminate oral biofilm. The mechanism of SiO 2 -P Ce6-IL against oral biofilm is showed in Figure 1.  [14], except the 2.4 mL of triethylamine and 2-bromoisobutyryl bromide (1.2 mL) were added to prepare SiO 2 -Br. The multiclawed SiO 2 -P Ce6-IL was prepared by ATRP. Briefly, 10 mg Ce6-IL and SiO 2 -Br dispersed into a mixture solution of ethanol and distilled water (5 : 1, V : V) for stirring 10 min at 800 rpm. 200 μL of PMDETA and 30 mg of CuCl were quickly transferred into the flask for reaction 8 h at 35°C. The SiO 2 -P Ce6-IL was washed with ethanol, distilled water.

Materials and Methods
The morphology of SiO 2 -P Ce6-IL was examined by TEM and SEM. The change of SiO 2 -P Ce6-IL zeta potentials in pH 4.5 and 7.4 was measured by a Delsa Nano C particle analyzer (Beckman Coulter Ireland Inc.).
The UV absorption of 5.0 mg·mL -1 SiO 2 -P Ce6-IL was measured by UV-vis (MAPADA). The Ce6 loading efficiency (L) was calculated as formula: L was SiO 2 -P Ce6-IL loading efficiency; C was the concentration of Ce6 (mg·mL -1 ); V was solution volume (mL); W was the weight of SiO 2 -P Ce6-IL (mg).

Journal of Nanomaterials
The Ce6 released in acidic microenvironment of oral biofilm (pH 4.5) was quantified by UV-vis. Briefly, 10 mg multiclawed SiO 2 -P Ce6-IL was placed in pH 4.5 solution 10 min and separated by centrifugation. The concentration of Ce6 in supernatant was examined by UV-vis.
DPBF was used to measure the generation of 1 O 2 [15]. Briefly, 2.0 mL DMSO solution containing SiO 2 -P Ce6-IL (equalled 100 μM Ce6) and 100 μM DPBF was irradiated by 660 nm light with a power density of 0.5 W·cm -2 . The absorbance of DPBF at about 410 nm was taken to record at 1 and 2 min.
2.3. Binding Ability of SiO 2 -P Ce6-IL to Oral Biofilm. Hydroxyapatite tablets (6.0 mm in diameter, 1.5 mm in thickness) were washed twice with PBS and incubated with artificial saliva to obtain saliva-coated hydroxyapatite. The S. mutans biofilm was formed on hydroxyapatite surfaces in BHIB medium which contained S. mutans (10 6 ) and 2% sucrose (w/v) for incubated 72 h at 37°C.
The binding capacity of SiO 2 -P Ce6-IL to the hydroxyapatite and S. mutans biofilm was measured by UV-vis. The 0.1 mM multiclawed SiO 2 -P Ce6-IL was incubated with hydroxyapatite and biofilm in pH 4.5 and 7.4 solutions for 10 min at 37°C, and then, the concentration of SiO 2 -P Ce6-IL was measured before and after adsorption to calculated binding capacity.
In order to examine the binding ability of SiO 2 -P Ce6-IL to oral biofilm, the interaction between SiO 2 -P Ce6-IL with S. mutans biofilm was measured by AFM. The procedure was as follows: the AFM tip (NP-O10, Bruker) contacted with epoxy. After waiting for 10 min, 1 μL SiO 2 -P Ce6-IL (0.1 mM) was put on the tip and heated to completely cure epoxy. Then, the AFM tip was put in ultrapure water and ultrasoni-cated for 30 s to get rid of unattached SiO 2 -P Ce6-IL . The SEM was used to check the AFM tip modified with SiO 2 -P Ce6-IL .

Photosensitizer Concentration in
Biofilm. The S. mutans biofilm was stained with Alexa Fluor 647. Briefly, 1 μM Alexa Fluor 647 was added to the S. mutans biofilms for 6 h. 20 μL of SiO 2 -P Ce6-IL or Ce6 (120 μM) was added onto the S. mutans biofilm interaction for 30 min, and then, wash with PBS 3 times to remove unconjugated SiO 2 -P Ce6-IL or Ce6. The laser scanning confocal microscopy (LSCM, Leica TCS SP8 STED 3X Super-resolution Confocal Microscope, Germany) was used to examine the concentration of Ce6 in biofilm.
2.5. Antibiofilm Activity. The S. mutans biofilm was treated with PBS, SiO 2 -P Ce6-IL , and Ce6. The biofilm incubated with BacLight live/dead dye in the dark after illumination for 15 min to observe dead/live bacteria. The CLSM images were taken to analyze the live and dead bacteria.
2.6. In Vivo Efficacy. Animal experiments were performed on a well-established model of dental caries disease as described in previous literatures [16]. Briefly, rats with two weeks old were purchased from animal center of the Air Force Medical University and screened for infection with S. mutans. Any rats infected with S. mutans prior to inoculation were removed. The animals were infected orally using an actively growing culture of S. mutans by oral swabbing three times a day for 2 weeks. Infected animals were randomly placed into three treatment groups of n = 5, and their teeth treated topically once daily. The treatment groups included (1)     5 Journal of Nanomaterials free Ce6 (0.1 mM Ce6) using a 660 nm LED light (0.5 W/ cm 2 ) for 15 min. Each group was provided the National Institutes of Health cariogenic diet 2000 and 5% sucrose water ad libitum. The experiment proceeded for 2 weeks; all animals were weighed weekly. At the end of the experimental period, animals were sacrificed, and the teeth were observed by SEM. Finally, the heart, liver, spleen, lung, and kidney were harvested for histological sections (H&E staining analysis on a Leica SP8 microscope).

Hemolysis Assay.
The red blood cells washed with PBS until the supernatant was pellucid and was diluted to 2 vol% by PBS. The Ce6 and SiO 2 -P Ce6-IL (0.1 mM) were immersed into 2 vol% red blood cell solutions (5 mL for each tube) and incubated at 37°C for 3 h, respectively. Then, the samples were centrifuged at 1500 rpm for 10 min, and the OD values were recorded at 545 nm. The red blood cells treated with water as the positive control, while the red blood cells treated with PBS as negative control. The hemolysis rate was determined by the equation (2): Besides hemolysis rate, the major organs including the hearts, livers, spleens, lungs, and kidneys were collected and stained with H&E for further evaluation of biocompatibility as previously reported [17].

Results and Discussion
3.1. Characterization of SiO 2 -P Ce6-IL . In order to strong interaction, the 8.92% Br of ATRP grafted on SiO 2 (Figure 2(d)). As showed in Figure 2(a), the size of SiO 2-P Ce6-IL was about 70 nm, and the surface was more rough after initiated by CuCl. Element analysis displayed that N elements of P Ce6-IL were on the SiO 2 . The location of Si   Journal of Nanomaterials and N elements was further analyzed by the Spherical Aberration Corrected Transmission Electron Microscope (ACTEM) analysis software. The analysis results showed that the N element was on the surface of Si (Figure 2(b)).
These results indicated that the P Ce6-IL was successful grafted on the surface of SiO 2 . Because 1 O 2 was a major factor in photodynamic therapy [18], the 1 O 2 production of SiO 2 -P Ce6-IL was examined   (Figure 2(c)). The SiO 2 -P Ce6-IL had higher 1 O 2 . More 1 O 2 production indicated that SiO 2 -P Ce6-IL had strong oxidation capacity and high PACT efficacy.

Binding
Ability of SiO 2 -P Ce6-IL with Oral Biofilm. The Streptococcus mutans (S. mutans) is one of the primary pathogenic bacteria that cause caries. In this work, S. mutans biofilm as model was used to study antibacterial ability of SiO 2 -P Ce6-IL . To mimic the bacterial biofilm on the teeth, bacteria were grown for 72 h on hydroxyapatite disks. Due to acid microenvironment of S. mutans biofilm, the Ce6 was protonated and released; then, SiO 2 -P Ce6-IL changed to positive charged SiO 2 -P IL + . The biofilm had anions such as carboxyl, phosphoryl, and glycerate at the proteins or polysaccharides would produce negatively charge [19]. The electrostatic interactions would occur between SiO 2 -P IL + and S. mutans biofilm. In order to detect interaction strength, the AFM was used to detect the interaction between SiO 2 -P IL + and S. mutans biofilm. As showed in Figure 3(a), the SiO 2 -P Ce6-IL has been successfully modified on the AFM probe. The interaction between SiO 2 -P Ce6-IL with S. mutans biofilm was 954.02 nN which contributed by imidazolium cation and dodecyl in the SiO 2 -P IL + . However, the interaction of Ce6 with S. mutans biofilm was only 23.35 nN, and this weak force was difficult to resist saliva cleaning. This strong interaction was also proved by SEM. As showed in Figure 4(c), the SiO 2 -P IL + like multiclawed octopus and could firmly adsorb on the S. mutans biofilm (EPS). Along with massive positive charges of SiO 2 -P IL + , the surface potential of S. mutans biofilm changed from -117 to 35 mV, and the adhesion of S. mutans biofilm was decreased from -76 nN to -25 nN. However, the adhesion of S. mutans biofilm treated by Ce6 alone was -45 nN. Due to the strong adhesion, the oral biofilm infection was difficult to eradicate. So, the decreased adhesion of SiO 2 -P Ce6-IL will be beneficial to eliminating biofilm infection.
3.3. Antibacterial Activity In Vitro. As a result of strong interaction, the SiO 2 -P IL + combined with biofilm firmly and the problem of saliva cleaning was solved. But the barrier of biofilm also severely restricted the PACT efficacy against oral biofilm. As showed in Figure 4(a), the blue was S. mutans biofilm, and the red Ce6 was difficult to enter the biofilm (Figure 4(a)). Limited by lifetime and diffusion distance of 1 O 2 [20], the Ce6 was difficult to damage protected bacteria outside biofilm. As showed in Figure 4(b), green represented live bacteria, and red represented dead bacteria. Most bacteria were alive after treated by Ce6 alone. So it is very important to destroy the dense structure of biofilm to improve the Ce6 concentration in biofilm. Compared to Ce6, SiO 2 -P Ce6-IL had hydrophobic dodecyl chain which like suckers on the arm of octopus and could destroy the structure of biofilm. In order to examine the membrane breaking effect of SiO 2 -P Ce6-IL , the morphology of S. mutans biofilm was characterized by SEM. As showed in Figure 4(c), the pores were created in the S. mutans biofilm. Once the biofilm was broken, the Ce6 was highly concentrated in biofilm (Figure 4(a)). After illumination for 15 min, most of S. mutans in the biofilm were killed by SiO 2 -P Ce6-IL (Figure 4(c)). The SiO 2 -P Ce6-IL greatly  3.4. In Vivo Antibiofilm Assessment. The in vitro results revealed that multiclawed SiO 2 -P Ce6-IL had excellent efficacy against S. mutans biofilms. To further verify these conclusions, the in vivo experiments were designed. The infected mice were divided into three groups: treated with PBS, Ce6 with illumination for 15 min, and SiO 2 -P Ce6-IL with illumination for 15 min. The therapeutic effects of SiO 2 -P Ce6-IL were evaluated by SEM. As showed in Figure 5, the treatment with SiO 2 -P Ce6-IL was very effective in preventing the development of dental caries and completely blocked extensive enamel damage. In contrast, treatment with Ce6 alone was no significant effect and resulted in enamel loss and damage, then led to dental caries.
3.5. Biocompatibility Evaluation. Although SiO 2 -P Ce6-IL could effective combine with oral biofilm and almost impossible to transport into the stomach from the mouth, it was important to evaluate their biocompatibility. So the cytotoxicity, hemolysis rate, and the histological section of main organs including the heart, liver, spleen and lung, and kidney were examined. The results show that no abnormal effects or damages were observed in these organs ( Figure 6), and the body weights of rats treated with SiO 2 -P Ce6-IL were similarly to healthy rats. The hemolysis rate and cytotoxicity were 4.2% (limit of clinical hemolysis rate < 5%) [21] and above 90% within therapeutic concentration ( Figure 6). The SiO 2 -P Ce6-IL was a potential safe material for clinical applications in oral biofilm infection.

Conclusions
In this work, the SiO 2 -P Ce6-IL with charge reversal and high binding ability was prepared. The potential measurements showed that SiO 2 -P Ce6-IL could turn into positive and combine with EPS firmly in the acidic microenvironment of oral biofilm. The morphology of S. mutans biofilm results showed that SiO 2 -P Ce6-IL could combine with EFS and destroy the structure of biofilm. The laser scanning confocal microscopy results found that the concentration of Ce6 in S. mutans biofilm was greatly improved, and most of bacteria in biofilm were killed. In vivo experiments showed that the SiO 2 -P Ce6-IL was a highly desirable property for oral biofilm infection and had high quality in the prevention of dental caries. Most importantly, SiO 2 -P Ce6-IL had good biocompatibility in therapeutic effective concentrations.

Data Availability
The experimental data used to support the findings of this study are included within the article.