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Antibacterial Efficacy of Photosensitizer Functionalized Biopolymeric Nanoparticles in the Presence of Tissue Inhibitors in Root Canal Annie Shrestha, BDS, MSc, and Anil Kishen, BDS, MDS, PhD Abstract Introduction: Application of antibacterial nanoparticles to improve root canal disinfection has received strong interest recently. The current study aims to assess the antibacterial effect of a novel photosensitizer (rose bengal functionalized chitosan nanoparticles [CSRBnp]) to eliminate bacteria in the presence of various root canal constituents that are known to inhibit the antibacterial efficacy of root canal disinfectants. Methods: The synthesized CSRBnp were evaluated for size, charge, and singlet oxygen release. The antibacterial effect of CSRBnp was tested on planktonic Enterococcus faecalis with or without pretreatment by using different inhibiting agents such as dentin, dentin-matrix, pulp tissue, bacterial lipopolysaccharides, and bovine serum albumin (BSA). Bacterial survival was assessed in a time-dependent manner. The antibacterial effects after photodynamic activation on CSRBnp, a cationic photosensitizer (methylene blue), and an anionic photosensitizer (rose bengal [RB]) in the presence of inhibitors were also evaluated. Results: CSRBnp were 60 20 nm in size and showed reduced rate of singlet oxygen release as compared with methylene blue and RB. Pulp and BSA inhibited the antibacterial effect of CSRBnp (without photoactivation) significantly (P < .05) even after 24 hours of interaction. In case of photodynamic therapy, the pulp and BSA significantly inhibited the antibacterial activity of all 3 photosensitizers. CSRBnp showed residual effect and completely eliminated the bacteria after 24 hours of interaction after photodynamic therapy. Conclusions: The inherent antibacterial activity of polycationic chitosan nanoparticles and the singlet oxygen released after photoactivation of RB synergistically provided CSRBnp the potential to achieve significant antibacterial efficacy even in the presence of tissue inhibitors within root canals. (J Endod 2014;40:566–570)
Key Words Antibacterial, chitosan, nanoparticles, tissue inhibitors
D
espite the high antimicrobial efficacy of conventional disinfectants in vitro, clinical studies have demonstrated bacterial persistence within the root canal system even after cleaning and shaping procedures (1, 2). The effectiveness of antimicrobial irrigants is known to be compromised under in vivo conditions because of the complex root canal anatomy that permits localization of bacteria in the inaccessible areas. In addition, the effectiveness of antibacterial chemicals depends on the concentration, time, and volume of irrigants inside the root canals. It is a challenging task to ensure optimum concentration/volume of irrigants for sufficient time in all locations of root canal system. The recent advances toward achieving predictable endodontic disinfection have focused on newer alternatives such as photodynamic therapy (PDT). Recent development in polycationic conjugates (3, 4) and nanoparticles (5, 6) has potentiated the antimicrobial effect of PDT. In this situation, it is highly pertinent to realize the importance of dentin constituents, tissue remnants, and serum products present within the root canals and their ability to neutralize the commonly used antibacterial disinfectants (7–9). Similar reduction in the antibacterial activities of newer disinfectants (chitosan nanoparticles and PDT) has also been reported (10). Considering the negative effects of tissue inhibitors on the currently available root canal disinfectants, it is important to develop antimicrobials that are effective even in their presence. Nanoparticles are insoluble particles that are no greater than 100 nm in size. The significantly increased surface area of nanoparticles contributes to their novel and improved physical, chemical, and biological properties (11). They could be used to deliver various antibacterial agents including photosensitizers. Nanoparticles that are based on metals or polymers are also being assessed for augmenting the current endodontic disinfection methods (5, 12). Coating or surface attachment of photosensitizers (eg, rose bengal [RB] or toluidine blue) to nano-sized glass beads or gold resulted in significantly improved antibacterial properties (13, 14). Chitosan, a bioactive polymer, offers an attractive material for conjugation with other reactive molecules because of the free amine and hydroxyl groups. This versatile polymer can be synthesized into nanoforms for various biomedical and pharmaceutical applications (15). Broad-spectrum antibacterial activity, biocompatibility, and ability to resist aging for longer periods provide antibacterial nanoparticles significant advantages in root canal disinfection (12, 16). Nevertheless, their antibacterial activity was found to be seriously compromised in the presence of various root canal constituents, mainly pulpal tissues and bovine serum albumin (BSA) (10). PDT is based on the use of a nontoxic photosensitizer, which, when activated by using a low energy light, results in the production of free radicals such as singlet oxygen (17). Singlet oxygen generated is highly reactive and is known to target various bacterial sites such as cell wall, nucleic acid, as well as membrane proteins (17, 18).
From the Discipline of Endodontics, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Anil Kishen, Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G 1G6. E-mail address:
[email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.09.013
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Basic Research—Technology The antibacterial activity of PDT was also compromised in the presence of root canal constituents such as pulpal tissues, serum, dentin matrix, and bacterial remnants (lipopolysaccharides) (10, 19). The degree of inhibition also depended on the class of photosensitizer used, either phenothiazines with positive charge (methylene blue [MB]) or xanthenes with negative charge (RB). The reduced efficacy of antibacterial PDT was mainly attributed to the interaction of photosensitizers with the tissue inhibitors, leading to reduced binding to the bacterial cell, reduced uptake into bacterial cells, and decreased half-life of the singlet oxygen produced on photoactivation (20, 21). The antibacterial effect of PDT was found to be significantly improved when chitosan was used along with PDT (22). This was mainly attributed to the membrane destabilizing/permeabilizing effect of chitosan that could subsequently enhance the effect of singlet oxygen on bacterial cells. Conjugating the commonly used photosensitizer with chitosan and further making them into nanosize offer an attractive single-step option for disinfection. The photosensitizer RB with free carboxyl groups forms chemical cross-link with the amine groups of chitosan nanoparticles (23). The RB functionalized chitosan nanoparticles (CSRBnp) are hypothesized to offer the following advantages: 1. Affinity to bacterial cells, interaction resulting in bacterial elimination by cationic chitosan (12) 2. Further increased interaction and uptake caused by the nano-sized particles (5) 3. Singlet oxygen released after photoactivation of RB (24) On the basis of the above hypothesis, CSRBnp were synthesized and characterized for size, charge, and ability to yield singlet oxygen, and their antibacterial efficacy was assessed in the presence of various tissue inhibitors with and without photoactivation.
Materials and Methods Chitosan, RB, MB, lipopolysaccharide (LPS) from Escherichia coli, and 1,3-diphenylisobenzofuran (DPBF) were purchased from Sigma Aldrich (St Louis, MO). The following agents were tested for the inhibitory effects: (1) 28 mg dentin powder; (2) 10 mg fresh bovine pulp, frozen and powdered; (3) 5 mg dentin-matrix; and (4) 2% and 18% BSA and LPS (1 mg/mL) (7, 10). Extracted human third molars and bovine teeth from the slaughterhouse were obtained after approval from the Research Ethics Office, University of Toronto. Dentin powder, dentinmatrix, and pulpal tissues were obtained as mentioned in previous study (10). Enterococcus faecalis was used as a test organism because it was found in high prevalence in the persistent and retreatment cases (25). E. faecalis ATCC 29212 was grown overnight on brain-heart infusion (BHI) broth (Bacto; DIFCO Laboratories, Franklin Lakes, NJ). The culture was centrifuged (4500 rpm, 10 m), washed twice in sterile deionized water, and adjusted spectrophotometrically to a cell density of approximately 108 colony-forming units (CFU) per mL (optical density, 0.7).
Synthesis and Characterization of CSRBnp CSRBnp were synthesized following the previous literature (4, 12). The synthesized CSRBnp were evaluated for their size by using transmission electron microscopy (TEM) and charge by using Zetasizer (Malvern Instruments Ltd, Malvern, UK). Photo-oxidative characterization was conducted to assess the singlet oxygen yield on photoactivation. Measurements were carried out in a 24-well plate according to a procedure described previously (4). Generation of singlet oxygen by photoactivation of CSRBnp 0.3mg/mL, RB 10 mmol/ L, and MB 10 mmol/L was studied photometrically by using DPBF, a singlet oxygen scavenger. Two milliliters DPBF (200 mmol/L) was added to 100 mL different PS solutions. A broad-spectrum Lumacare JOE — Volume 40, Number 4, April 2014
(LumaCare Inc, Newport Beach, CA) lamp fitted with a 540- or 660-nm filtered fiber was used as a light source. The decrease in absorbance intensity at 410 nm was monitored as a function of time by using a UV-Visible microplate reader (Epoch; Biotek, Winooski, VT) and was proportional to the rate of singlet oxygen production.
Time-dependent Evaluation of Antibacterial Activity in the Presence of Inhibitors The experimental inhibitors were added into 1 mL of the 0.3 mg CSRBnp and incubated in sealed test tubes at 37 C for 1 hour (7, 10). This concentration of CSRBnp was chosen on the basis of the characterization experiments conducted in our laboratory. The control group included CSRBnp without any inhibitors. After 1 hour of incubation, the CSRBnp and inhibitor solution were added to the bacterial cell pellets and vortexed to ensure uniform interaction. The antibacterial activity was monitored at different time intervals by taking 50-mL samples of bacterial suspension that were serially diluted and plated onto freshly poured BHI agar plates and incubated for 24 hours at 37 C to determine the CFU of viable bacteria. Effect on Antibacterial Photodynamic Activity in the Presence of Inhibitors In case of PDT, the photosensitizers (CSRBnp, RB, and MB) were preincubated with 2 experimental inhibitors (pulpal remnants and BSA) that showed highest inhibitory effect (4, 26). The photosensitizers and inhibitors mixture was then added into the cell pellet of E. faecalis and photosensitized for 15 minutes in the dark. Then the bacterial cells were centrifuged to remove the unbound photosensitizers and subjected for PDT at 5 and 10 J/cm2 energy dose for 1.66 and 3.33 minutes, respectively. The samples were evaluated immediately after PDT as well as after further interaction for 24 hours. The samples were serially diluted, and various dilutions were plated onto freshly poured BHI agar plates for 24 hours at 37 C. Statistical Analysis The experiments were carried out in triplicates (CFU counts) with 3 samples per group each time, a total of 9 observations per treatment. Data values were transformed into percentage survival to reduce variance heterogeneity. Statistical analysis of E. faecalis percentage survival rates under the different inhibitor treatment conditions was performed by using one-way analysis of variance and Tukey multiple comparison test. A P value < .05 was considered to indicate statistical significance.
Results Synthesis and Characterization of CSRBnp Figure 1A shows the spherical aggregates of CSRBnp under the TEM with smooth surface and in the 60 20 nm size range. The charge of CSRBnp was positive, 30 0.06 mV. These average values were obtained from 6 independent measurements of nanoparticles. CSRBnp produced singlet oxygen on photoactivation similar to RB, as observed by the decrease in the DPBF concentration (Fig. 1B). RB and MB showed rapid release of singlet oxygen. Time-dependent Evaluation of Antibacterial Activity in the Presence of Inhibitors CSRBnp showed complete killing of E. faecalis in the absence of inhibitors after 24-hour interaction (Fig. 2). Presence of dentin and LPS reduced the rate of antibacterial efficacy in the initial hours of interaction (1 and 8), and complete elimination was obtained after 24 hours Photosensitizer Functionalized Biopolymeric Nanoparticles
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Figure 1. (A) TEM image of CSRBnp. (B) Graph shows decrease in absorbance of DPBF because of singlet oxygen release after photoactivation of CSRBnp, RB, and MB.
(P < .05). The pulpal tissues showed highest inhibitory effect, followed by BSA with only 65% bacterial reduction even after 24 hours (P < .05).
Photodynamic Effect on Antibacterial Activity in the Presence of Inhibitors E. faecalis was completely eliminated with all the photosensitizers, CSRBnp, RB, and MB, after PDT in the absence of any inhibitors (Fig. 3) (P < .01). BSA and pulp inhibited the PDT mediated antibacterial activity of all 3 photosensitizers. The 24-hour interaction of CSRBnp after PDT resulted in complete elimination of bacteria even in the presence of inhibitors (Fig. 3A) (P < .01). The CSRBnp interaction without PDT also showed 50%–65% bacterial reduction. This post-PDT complete reduction of bacterial viability was not seen in cases of RB and MB. MB in the presence of BSA and RB in the presence of pulp showed increased killing after PDT and 24-hour interaction.
Discussion The chitosan nanoparticles are highly reactive cationic nanoscaled particles that interact physicochemically with other charged particles as well as bacteria. Furthermore, as compared with the microparticles of polymeric chitosan (23), the nanoparticles provide more surface area for binding of the photosensitizer, making it more reactive. The cationicity and the quantum size effect of nanoparticles could be further supplemented with singlet oxygen release in the presence of a photosensitizer. Conjugation of chitosan nanoparticles with RB could 568
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Figure 2. Killing of E. faecalis ATCC 29212 by CSRBnp in a time-dependent manner (A) in the presence of pulp, dentin, and dentin matrix and (B) LPS and BSA.
provide the combined activity of CSnp (bioactivity, quantum size effect, high affinity to bacterial cells, and biocompatibility and photosensitizer) (singlet oxygen release), thereby potentiating the antibacterial efficacy. CSRBnp possessed strong antibacterial properties against E. faecalis in a time-dependent manner, which is further enhanced on photoactivation. This could be mainly attributed to the affinity of chitosan nanoparticles toward bacterial cell surface and its ability to permeabilize cell wall combined with the singlet oxygen produced by PDT (12). Presence of charged particles in a solution could negatively impact the interaction and uptake of nanoparticles or photosensitizers into the bacterial cell, which in turn compromised the antibacterial efficacy. An infected root canal system will invariably contain necrotic pulpal tissue, bacterial by-products, and dentin debris after instrumentation. BSA has been used as a substitute for albumin, which is the main protein in human serum and inflammatory exudates (27). Purulent infected root canals may present with periapical tissue exudates in the apical portions of the root canal (28). BSA has also been used as a singlet oxygen quencher (29). Pulp tissues consist mainly of organic material such as cells and extracellular matrix proteins. These organic materials and dentin could act as a buffer against most commonly used root canal irrigants and medicaments (7, 30). Other than the direct electrostatic interaction of the charged nanoparticles with these inhibitors, the singlet oxygen produced after photoactivation could also react with JOE — Volume 40, Number 4, April 2014
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Figure 3. Killing of E. faecalis ATCC 29212 by CSRBnp and PDT by using (A) CSRBnp, (B) MB, and (C) RB in the presence of pulp and BSA. Bacterial survival was assessed immediately after PDT and after 24 hours of post-PDT treatment to evaluate residual activity.
these components (20). The singlet oxygen is the main cytotoxic agent in PDT, which induces oxidation of the biological substrates. The singlet oxygen interacts with various cell systems in a nonspecific manner, and its half-life is known to be diminished in the presence of serum (21). The CSRBnp showed the best ability to eliminate bacteria even in the presence of BSA and pulp, which further highlighted the advantage of JOE — Volume 40, Number 4, April 2014
combining the activity of PDT with bioactive photosensitizer functionalized nanoparticles. The antibacterial treatment that can retain its efficacy in the presence of such inhibitors would provide an attractive alternative in endodontic disinfection. Pulp and BSA strongly inhibited the antibacterial activity in both time-dependent and PDT manner. The immediate antibacterial activity of CSRBnp after PDT in the presence of BSA and pulp was not significant and was similar to the conventional PDT with MB and RB. On prolonged interaction, CSRBnp were able to reduce the bacteria up to 65% even without photoactivation and completely eliminated (100%) bacteria after photoactivation. The bacteria with minor insult after PDT may recover if environment is conducive for growth. Further reduction of bacterial numbers as seen with MB and RB in the presence of BSA and pulp, respectively, could be due to the inability of bacteria to grow that had undergone some damage because of the singlet oxygen. The presence of CSnp in the suspension of bacteria after PDT enhanced antibacterial activity of CSRBnp, as seen in the present study. Dentin, dentin-matrix, and LPS did not affect the antibacterial efficacy of CSRBnp. This could be mainly due to the weaker interaction of these inhibitors with CSRBnp, as shown previously in case of CSnp (10). The rate of singlet oxygen production by CSRBnp was lower when compared with the parent photosensitizer RB. This difference in singlet oxygen production is mainly due to the binding of RB to the polymeric CSnp. Guo et al (31) have also shown that when RB was embedded on the surface of silica nanoparticles, the singlet oxygen yield was slower. In addition, CS is known to scavenge oxygen, resulting in reduced singlet oxygen efficacy (32). Because the half-life of singlet oxygen is compromised in the presence of tissue fluids, prolonging the yield of singlet oxygen could be considered advantageous. The slow release of singlet oxygen could also be helpful in hypoxic root canal environment, because this will allow molecular oxygen replenishment during fractionation of PDT dosage (33). Thus, the enhanced antibacterial effect of CSRBnp was a combined effect of CSnp and singlet oxygen yield. Previously it has been shown that chitosan infiltrated and coated dentin collagen surface after photodynamic cross-linking process (34). The cross-linked and chitosan-infiltrated dentin collagen demonstrated improved mechanical properties and stability against enzymatic degradation. The dentin surface treated with chitosan nanoparticles after different root canal irrigants resulted in higher reduction of bacterial adherence (35). Similarly, CSRBnp in the root canal treatment could be applied to treat dentin surface that could prevent bacterial adhesion, thereby preventing bacterial recolonization and biofilm formation. However, the necessity of obtaining a well-designed consistent light delivery into the root canals presents as an issue. Optical fiber could be modified by tapering the end or by using diffuser to deliver light inside the root canals (36, 37). Use of a liquid conduit to uniformly irradiate root dentin has also been reported (24). The higher affinity of cationic chitosan antibacterial nanoparticles to bacterial cell surfaces and singlet oxygen release after photoactivation of RB provided a synergistic mechanism for CSRBnp to exert their antibacterial efficacy even in the presence of tissue inhibitors. In conclusion, CSRBnp are a novel antibacterial agent with distinct and potential benefits in root canal disinfection.
Acknowledgments Supported by the University of Toronto Start-up fund. The authors deny any conflicts of interest related to this study.
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