Basic Research—Technology
Photoactivated Polycationic Bioactive Chitosan Nanoparticles Inactivate Bacterial Endotoxins Annie Shrestha, BDS, MSc, PhD,* Martha Cordova, BSc, MSc,† and Anil Kishen, BDS, MDS, PhD* Abstract Introduction: The current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). Methods: Antimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a timedependent interaction with/without photoactivation (20 and 40 J/cm2). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide– and enzymelinked immunosorbent assay (tumor necrosis factor a and interleukin-6 expression)–based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. Results: CSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P < .005) even at 30 minutes of exposure. CSRBnps and MB with/without photoactivation significantly inactivated LPSs with reduced nitric oxide and tumor necrosis factor a expression (P < .05). Cell death and detachment after Ca(OH)2 treatment resulted in complete absence of all 3 inflammatory markers. Conclusions: Photodynamically activated CSRBnps caused significant inactivation of endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins. (J Endod 2015;-:1–6)
Key Words Chitosan, endotoxins, inactivation, nanoparticles, photodynamic
A
n effective endodontic disinfection necessitates the elimination of bacterial biofilms and the inactivation of their by-products to facilitate optimal periradicular tissue healing (1). A chemomechanical approach that combines mechanical instrumentation and chemical irrigation has been shown to be reasonably effective in lowering viable bacterial loads in root canals (2, 3). Nevertheless, conventional protocols fall short in the elimination/inactivation of bacterial modulins such as endotoxins/ lipopolysaccharides (LPSs) from infected root dentin (4). LPSs have been associated with endodontic infections with aggravated clinical symptoms (5, 6). The continuous leakage of LPSs from the infected root canal system and its accumulation in the surrounding periradicular tissues have been shown in an animal model (6). Current topical antibacterials in endodontics such as 2.5% sodium hypochlorite and 2% chlorhexidine gel produced only an incomplete elimination of LPSs in vivo (4). The inability of the current topical disinfectants and medications to neutralize LPSs may be associated with the persistence of apical periodontitis in an endodontically treated tooth (4, 7). Furthermore, the existence of LPSs within root dentin and dentinal tubules (7) may negatively impact the possibility of biologically based treatment procedures in endodontics. A newer antimicrobial is essential that can overcome the current limitations of eliminating both bacteria and endotoxins from infected root dentin. LPSs are released from the cell wall of gram-negative bacteria, the predominant species involved in primary endodontic infections (5, 8). They possess a strong negative charge because of their lipid moiety, which could be neutralized and sequestered with positively charged molecules (9). Calcium hydroxide (Ca[OH]2) relies on the sustained establishment of a high pH in the root canal system. It requires a longer treatment period for the effective inactivation of LPSs (10). Although in vitro studies have supported the use of Ca(OH)2 in neutralizing LPSs (11, 12), an in vivo study conducted by Vianna et al (13) reported that LPSs remained in the root canal system even after the application of Ca(OH)2 dressing for 7 days. It is suggested that the clinical effectiveness of Ca(OH)2 may be hindered by the lack of hydroxyl ion delivery through dentinal tubules, complex canal anatomy, and the buffering capacity of dentin (10). Recently, photodynamic therapy (PDT), which applies a low-energy light with a photosensitizer, has been shown to have the potential to enhance the disinfection efficacy of the conventional chemomechanical preparation (14, 15). PDT is also effective in inactivating the virulence factors from gram-negative bacteria and reducing the inflammatory potential of LPSs adherent to a titanium implant (16, 17). Cationic nanoparticle–conjugated photosensitizers have been shown to potentiate the antibacterial efficacy of PDT (15, 18, 19). Bioactive nanoparticles of chitosan and rose bengal–conjugated nanoparticles (CSRBnps) have shown significant antibiofilm activity against both single-species and multispecies bacterial biofilms, even in the
From the *Discipline of Endodontics and †Dental Research Institute, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Anil Kishen, Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.12.007
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Basic Research—Technology presence of inhibitors such as dentin powder and organic remnants (15, 20, 21). Chitosan particles and their modifications have also been shown to be effective in neutralizing LPSs (22, 23). Combining the binding affinity of polycationic chitosan nanoparticles and the effect of singlet oxygen release by photoactivated photosensitizers, we hypothesized that CSRBnps will effectively neutralize bacterial LPSs, thereby reducing the expression of inflammatory cytokines from macrophage cells. Thus, the aim of this study was to evaluate the ability of photoactivated CSRBnps in the inactivation of bacterial endotoxins and direct toxicity to macrophage cells.
Materials and Methods Ultrapure LPSs from Pseudomonas aeruginosa (Sigma-Aldrich, St Louis, MO) (endotoxin level 500,000 EU/mg) were used in the study; 1 mg/mL LPSs was prepared as a stock solution. Low-molecular-weight, low-viscosity chitosan particles (Sigma-Aldrich) were used for the synthesis of chitosan nanoparticles (CSnps) (24) and CSRBnps following previous literature (15). All other chemicals were pyrogen free with the highest analytic grade unless specified. CSnps (10 mg/mL), Ca(OH)2 (10 mg/mL), CSRBnps (0.3 mg/mL), and methylene blue (MB) (10 mmol/L) were prepared fresh in culture medium. The noncoherent light source (Lumacare-LC-122M; LumaCare, Newport Beach, CA) with interchangeable fiber bundles and 30-nm band-pass filters at ranges 660 15 nm and 540 15 nm for MB and CSRBnps, respectively, was used.
Cell Culture RAW 264.7 macrophage cells at passages 3 to 5 (1 106 cells/ml) were used in the experiment. Cells were added into each well of a sterile 24-well plate (BD Falcon, Franklin Lakes, NJ) and incubated for 24 hours (37 C, 5% CO2) to allow cell attachment. Nonadherent cells were removed, and the remaining cells were washed with medium. Neutralization Treatment of LPS LPSs (500 EU/mL) were subjected to the following treatment groups: Ca(OH)2, CSnps, MB, and CSRBnps. The time-dependent effect on treatment with Ca(OH)2 and CSnps was evaluated at 6 and 12 hours (37 C) (22). Both time-dependent (15 minutes and 6 hours) and photoactivated (20 and 40 J/cm2, time: 4 and 8 minutes, respectively) effects were evaluated for MB and CSRBnps at the 2 incubation periods. In the negative control group, the culture medium was without LPSs (LPS ), and in the positive control group (LPS+), LPSs were added in culture medium, which was then added into the cells. The treated and untreated LPSs from different groups were then added into cells, and the plates were incubated (37 C, 5% CO2) for 12 hours. The cell-free supernatants were collected by centrifugation at 10,000 rpm for 5 minutes (4oC) and stored at 20 C for further experiments. Measurement of Nitric Oxide The levels of nitric oxide (NO) in the cell culture supernatants were quantified by measuring the nitrite using the Griess Reaction System (Promega, Madison, WI) according to the manufacturer’s instructions. The NO concentration was determined quantitatively using the standard curve of nitrite. Measurement of Tumor Necrosis Factor a and Interleukin 6 Tumor necrosis factor a (TNF-a) and interleukin (IL)-6 concentrations released by cells in culture medium were determined using an 2
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enzyme-linked immunosorbent assay with the Quantikine Human TNFa and IL-6 Immunoassay Kit (R&D Systems, Minneapolis, MN). Purified recombinant TNF-a and IL-6 were used as the standard. TNF-a and IL-6 were expressed as concentration per milliliter based on the standard curve.
Cell Viability Assessment The cells were assessed for viability following treatment with Ca(OH)2, CSnps, MB, and CSRBnps. Ca(OH)2 and CSnps for were measured for their time-dependent effect at 30 minutes and 12 hours (37 C). The effect of MB and CSRBnps on cells after photosensitization (15 minutes, 37 C) (dark toxicity) and photoactivation (20 and 40 J/ cm2) was evaluated. The control groups were tested without any treatment. Cell viability expressed as percentage survival was determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cells after treatment were examined under a light microscope to assess the direct effect of treatment on cells. The cells were washed after the treatment period and stained using 0.5% trypan blue for 5 minutes. The cells were viewed under a light microscope (Axio Vert.A1; Zeiss, Jena, Germany) at 20 magnification. Statistical Analysis All experiments were repeated 3 times in triplicate. The statistical significance was analyzed by 1-way analysis of variance and the Tukey post hoc test. The significance was set at 0.05.
Results NO Release The NO release by macrophage cells after treatment with or without LPSs was significantly different (Fig. 1A). The Ca(OH)2-treated groups showed a similar NO release to that of the LPS-negative group (11–12 mmol/L). CSnps showed a reduction of NO production compared with the LPS-positive group (P < .05). However, compared with the LPS-negative group, the NO release was twice as high in the CSnp groups (P < .005) at both time points. CSRBnps without photoactivation (0 J/cm2) showed a time-dependent neutralization effect that reduced the NO release significantly at 6 hours when compared with 15 minutes of treatment with LPSs (P < .05) (Fig. 1B). At both time points, the photoactivation resulted in significantly reduced NO release, which was comparable with the LPS-negative group. The MB group at both time points showed significantly reduced NO release from the cells (P < .05). Photoactivation of MB did not produce further inactivation of LPSs. TNF-a and IL-6 Release TNF-a and IL-6 release was completely absent in the absence of LPSs. Ca(OH)2 showed no TNF-a and IL-6 release from cells. The CSnp group showed a decrease in TNF-a release without any significant difference from the LPS-positive group (Fig. 2A). However, CSnps after 12 hours of interaction with LPSs significantly reduced IL-6 release (P < .05) (Fig. 2C). Both the CSRBnp and MB groups showed significantly better inhibition of TNF-a release at 15 minutes (P < .05) compared with 6 hours of interaction with and without photoactivation (Fig. 2B and D). At 6 hours, both the CSRBnp and MB groups showed TNF-a reduction only after photoactivation. IL-6 release was reduced significantly (P < .05) with CSRBnps at both time points without photoactivation. Photoactivation (40 J/cm2) of CSRBnps resulted in a highly significant (P < .005) reduction of IL-6 expression by the macrophage cells. The treatment with MB did not produce any significant reduction of IL-6 release. JOE — Volume -, Number -, - 2015
Basic Research—Technology Cell Viability Figures 1C and D and 3 show the effect of various treatment groups on the macrophage cells using a mitochondrial activity assay and light microscopy. CSnps were found to be nontoxic with 100% cell viability for up to 12 hours of incubation (Fig. 1C). Ca(OH)2 was highly cytotoxic to macrophage cells at the given concentration, which resulted in 70% cell death within 30 minutes. CSRBnps with or without photoactivation did not exhibit any toxicity. Although photoactivation with MB resulted in the reduction of cell survival, this change was not statistically significant (P = .23). The light microscopic images showed few dead cells attached on the plates of the Ca(OH)2 group (Fig. 3A). The trypan blue exclusion assay confirmed the presence of live and well-attached cells in all other treatment groups (Fig. 3B–D).
Discussion Gram-negative, aerobic bacteria such as Pseudomonas species have been associated with chronic infections and have been detected in persistent root canal infections (25, 26). The influence of LPSs from P. aeruginosa on macrophage cells was found to be significantly higher compared with that of Porphyromonas gingivalis in our preliminary experiments (data not shown). Endotoxins present in the root canals as well as within dentinal tubules could be contributed to persistent endodontic infection because they may act as a reservoir of continuous irritation to the periapical tissues (1, 4, 27). The high endotoxin concentration of 500 EU/mL used in this study correlated with endotoxins found in symptomatic root canal infections with pulp necrosis and apical periodontitis (28). Intracanal causes of the host immune response during periapical inflammation include the immune cells, intracellular mediators, effector molecules, and humoral antibodies (29). The presence of macrophages in human inflammatory periapical lesions has been long recognized. Neutrophils are known to dominate during the acute phase of the disease, and lymphocytes, macrophages, and plasma cells accumulate in the chronic phase of the disease (29). In chronic infection, macrophages are the primary innate immune cells to respond to invading microorganism. They play an important role in recognizing the molecular patterns on bacterial cells, initiating internalization and killing. Chronic infections are manifested by a cascade of immune reactions involving the generation of several cytokines, such as TNF-a, IL-1, IL-6, IL-8, effector molecules (eg, matrix metalloproteinase), NO, reactive oxygen species, and antibodies (30). TNF-a and IL-6 are considered to be the 2 major cytokines produced by the activated macrophages (29). The current study showed that PDT using CSRBnps and MB were effective in neutralizing LPSs, subsequently reducing the inflammatory potential of LPSs. Ca(OH)2 produced high cytotoxicity upon direct contact with the macrophage cells. The detachment and removal of the cells from plates resulted in the absence of any NO, TNF-a, and IL-6 production in the Ca(OH)2 group. The stand-alone effect of CSnps was not sufficient to reduce the LPS-induced inflammatory potential (NO and TNF-a) significantly. They only showed a reduction of IL-6 after 12 hours of interaction. Figure 1. NO release by macrophage cells stimulated with LPSs and treated with (A) Ca(OH)2 and CSnps and (B) CSRBnp and MB treatment with and without photoactivation. LPSs were allowed to react with CSRBnps and MB for 15 minutes and 6 hours before photoactivation. NO release in the LPS+ group was 27 mmol/L, and in the LPS group, it was 12 mmol/L. A cytotoxicity assay to assess the effect of (C) Ca(OH)2 and CSnps in a timedependent manner and (D) CSRBnps and MB after photoactivation of macrophage cells in vitro.
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Basic Research—Technology MB with/without photoactivation resulted in a slight decrease in cell viability, yet it produced a significant reduction of NO and TNF-a. However, no effect was observed on IL-6 production from activated macrophage cells. MB has been shown to form stable metachromatic complexes with LPSs of P. aeruginosa and Escherichia coli (31). The formation of MB dimers in the presence of LPSs could have aggregated and made LPSs unavailable to induce inflammatory mediators from macrophage cells. CSRBnps significantly neutralized LPSs after photoactivation. NO and IL-6 were reduced significantly even without photoactivation, whereas the inactivation of TNF-a required photoactivation. The effect of CSRBnps on reducing NO production without photoactivation was time dependent. This could be caused by the of LPS-CSRBnp complexes that start being formed in the initial phase of interaction (15 minutes) and are more stable after 6 hours of interaction. At 15 minutes, there could be LPSs that are either neutralized or still active, which result in variance in the production of NO from macrophages (Fig. 1B). The production of NO, IL-6, and TNF-a by macrophages has been used to determine the inflammatory potential of LPSs (12, 32). The neutralization of LPSs with CSRBnps and MB resulted in a decreased production of inflammatory cytokines in the present study. Marton and Kiss (32) reported an altered balance in the production and elimination of reactive oxygen intermediates in chronic apical periodontitis. TNF-a has a direct cytotoxic effect and a general debilitating effect in chronic disease. Macrophage-derived TNF-a has numerous systemic and local effects (30). The presence of TNF-a has been reported in human apical periodontitis lesions and in the root canal exudates of teeth with apical periodontitis. NO is synthesized by a complex family of enzymes called NO synthases. NO is reactive nitrogen intermediate and one of the initial and nonspecific responses to infection by macrophages. NO synthesis is increased in periapical infection as a result of macrophage infiltration in the periapical tissues (33). Both CSRBnps and MB were more effective in reducing NO expression in the present study. NO plays an important role in the pathogenesis of periapical lesions either by directly or indirectly modulating the production of other proinflammatory cytokines such as IL-6 and TNF-a. These cytokines are highly potent inflammatory markers, and only CSRBnps were able to significantly inhibit IL-6 and TNF-a expression. However, the present study is not able to explain the higher efficacy of CSRBnps without photoactivation to reduce IL-6 expression compared with TNF-a. Studies to understand the effect of the LPS structure on the expression of specific cytokines are needed to explain this observation. Chitosan is a polycation that possesses a positive charge in each of its glucosamine units (34). They formed stable complexes with negatively charged bacterial endotoxins, thereby reducing the inflammatory potential of activated macrophages (23). However, care should be taken to use chitosan of low molecular weight to avoid gelation caused by charge interaction. In this study, we used a lowmolecular-weight, low-viscosity chitosan to synthesize nanoparticles and photosensitizer-conjugated nanoparticles (20). CSnps alone were not as effective as CSRBnps toward LPS neutralization. The LPS neutralization effect of CSRBnps may be attributed to the smaller size of CSRBnps (60 nm) compared with CSnps (80 nm) (ie, a positive
Figure 2. (A and B) TNF-a and (C and D) IL-6 secretion by macrophage cells stimulated with LPSs and treated with Ca(OH)2, CSnps, CSRBnps, and MB. The interaction of LPSs with Ca(OH)2 and CSnps was assessed with time, and CSRBnps and MB were assessed with and without photoactivation. TNF-a expression in the LPS+ group was 5.14 ng/mL, and in the LPS group, it was 0.02 ng/mL. IL-6 expression in the LPS+ group was 795 pg/mL, and in the LPS group, it was 0.09 pg/mL.
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Figure 3. Light microscopic images of macrophage cells after direct contact with (A) Ca(OH)2, (B) CSnps, (C) CSRBnps, and (D) MB. The cells were stained with trypan blue stain to assess the cell morphology and membrane damage after the initial 30 minutes of treatment. The black arrows show dead cells or disrupted cell mass with trypan blue uptake.
charge [15] with a high affinity toward LPSs). The singlet oxygen produced upon photoactivation might further disintegrate the lipid moiety of LPSs. The PDT parameters were chosen based on previous studies that resulted in complete elimination of bacterial biofilms in root canal models (15, 18). Burns et al (9) have reported that small molecules with 2 protonable cationic groups effectively bind and neutralize endotoxins by interacting with the phosphate moieties of the lipid A component (9). PDT has shown promising results in eliminating endodontic bacteria/biofilms (14, 15), yet investigations examining its effect on LPSs are relatively limited (17). In the current study, photoactivation of CSRBnps resulted in significant neutralization of endotoxins even at a high concentration. CSRBnps have been previously shown to exhibit significant antibiofilm efficacy (15), stabilize dentin collagen after photo– cross-linking, and inhibit collagenolytic activity (35). Furthermore, the importance of selecting agents that could selectively inactivate LPSs while sparing the mammalian cells is highly beneficial in a clinical scenario in which treatment of infected tissue is in close proximity to normal host tissues (intraradicular and extraradicular regions close to the apical foramen). Considering these findings, CSRBnps could serve as an efficient antimicrobial agent for endodontic application.
Acknowledgments Supported by the American Association of Endodontists Foundation, Research Grant Program (Fund #494223), University of Toronto (Fund #928133), and Canadian Foundation of Innovation (Fund #493891). The authors deny any conflicts of interest related to this study.
References 1. Nair PN. On the causes of persistent apical periodontitis: a review. Int Endod J 2006; 39:249–81.
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2. Nair PN, Henry S, Cano V, Vera J. Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after ‘‘one-visit’’ endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99:231–52. 3. Siqueira JF Jr, Alves FR, Versiani MA, et al. Correlative bacteriologic and microcomputed tomographic analysis of mandibular molar mesial canals prepared by self-adjusting file, reciproc, and twisted file systems. J Endod 2013;39:1044–50. 4. Gomes BP, Martinho FC, Vianna ME. Comparison of 2.5% sodium hypochlorite and 2% chlorhexidine gel on bacterial lipopolysaccharide reduction from primarily infected root canals. J Endod 2009;35:1350–3. 5. Schein B, Schilder H. Endotoxin content in endodontically involved teeth. J Endod 1975;1:19–21. 6. Yamasaki M, Nakane A, Kumazawa M, et al. Endotoxin and gram-negative bacteria in the rat periapical lesions. J Endod 1992;18:501–4. 7. Horiba N, Maekawa Y, Matsumoto T, Nakamura H. A study of the distribution of endotoxin in the dentinal wall of infected root canals. J Endod 1990;16:331–4. 8. Fabricius L, Dahlen G, Sundqvist G, et al. Influence of residual bacteria on periapical tissue healing after chemomechanical treatment and root filling of experimentally infected monkey teeth. Eur J Oral Sci 2006;114:278–85. 9. Burns MR, Jenkins SA, Wood SJ, et al. Structure-activity relationships in lipopolysaccharide neutralizers: design, synthesis, and biological evaluation of a 540membered amphipathic bisamide library. J Comb Chem 2006;8:32–43. 10. Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide: a critical review. Int Endod J 1999;32:361–9. 11. Safavi KE, Nichols FC. Alteration of biological properties of bacterial lipopolysaccharide by calcium hydroxide treatment. J Endod 1994;20:127–9. 12. Barthel CR, Levin LG, Reisner HM, Trope M. TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS. Int Endod J 1997;30:155–9. 13. Vianna ME, Horz HP, Conrads G, et al. Effect of root canal procedures on endotoxins and endodontic pathogens. Oral Microbiol Immunol 2007;22:411–8. 14. George S, Kishen A. Advanced noninvasive light-activated disinfection: assessment of cytotoxicity on fibroblast versus antimicrobial activity against Enterococcus faecalis. J Endod 2007;33:599–602. 15. Shrestha A, Hamblin MR, Kishen A. Photoactivated rose bengal functionalized chitosan nanoparticles produce antibacterial/biofilm activity and stabilize dentincollagen. Nanomedicine 2014;10:491–501. 16. Giannelli M, Pini A, Formigli L, Bani D. Comparative in vitro study among the effects of different laser and LED irradiation protocols and conventional chlorhexidine treatment for deactivation of bacterial lipopolysaccharide adherent to titanium surface. Photomed Laser Surg 2011;29:573–80.
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Basic Research—Technology 17. Komerik N, Wilson M, Poole S. The effect of photodynamic action on two virulence factors of gram-negative bacteria. Photochem Photobiol 2000;72:676–80. 18. Shrestha A, Kishen A. Polycationic chitosan conjugated photosensitizer for antibacterial photodynamic therapy. Photochem Photobiol 2012;88:577–83. 19. Pagonis TC, Chen J, Fontana CR, et al. Nanoparticle-based endodontic antimicrobial photodynamic therapy. J Endod 2010;36:322–8. 20. Shrestha A, Kishen A. Antibiofilm efficacy of photosensitizer-functionalized bioactive nanoparticles on multispecies biofilm. J Endod 2014;40:1604–10. 21. Shrestha A, Kishen A. Antibacterial efficacy of photosensitizer functionalized biopolymeric nanoparticles in the presence of tissue inhibitors in root canal. J Endod 2014;40:566–70. 22. Yoon HJ, Moon ME, Park HS, et al. Chitosan oligosaccharide (COS) inhibits LPSinduced inflammatory effects in RAW 264.7 macrophage cells. Biochem Biophys Res Commun 2007;358:954–9. 23. Yermak IM, Davidova VN, Gorbach VI, et al. Forming and immunological properties of some lipopolysaccharide-chitosan complexes. Biochimie 2006;88: 23–30. 24. Shrestha A, Shi Z, Neoh KG, Kishen A. Nanoparticulates for antibiofilm treatment and effect of aging on its antibacterial activity. J Endod 2010;36:1030–5. 25. Siren EK, Haapasalo MP, Ranta K, et al. Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation. Int Endod J 1997;30:91–5.
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26. Cheung GS, Ho MW. Microbial flora of root canal-treated teeth associated with asymptomatic periapical radiolucent lesions. Oral Microbiol Immunol 2001;16: 332–7. 27. Marton IJ, Kiss C. Protective and destructive immune reactions in apical periodontitis. Oral Microbiol Immunol 2000;15:139–50. 28. Martinho FC, Gomes BP. Quantification of endotoxins and cultivable bacteria in root canal infection before and after chemomechanical preparation with 2.5% sodium hypochlorite. J Endod 2008;34:268–72. 29. Nair PN. Apical periodontitis: a dynamic encounter between root canal infection and host response. Periodontol 2000 1997;13:121–48. 30. Tracey KJ. Tumour Necrosis Factor-alpha. London: Academic Press; 1994. 31. Usacheva MN, Teichert MC, Biel MA. The interaction of lipopolysaccharides with phenothiazine dyes. Lasers Surg Med 2003;33:311–9. 32. Marton IJ, Kiss C. Characterization of inflammatory cell infiltrate in dental periapical lesions. Int Endod J 1993;26:131–6. 33. Brennan PA, Thomas GJ, Langdon JD. The role of nitric oxide in oral diseases. Arch Oral Biol 2003;48:93–100. 34. Kumar MN, Muzzarelli RA, Muzzarelli C, et al. Chitosan chemistry and pharmaceutical perspectives. Chem Rev 2004;104:6017–84. 35. Persadmehr A, Torneck CD, Cvitkovitch DG, et al. Bioactive chitosan nanoparticles and photodynamic therapy inhibit collagen degradation in vitro. J Endod 2014;40: 703–9.
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Photoactivated Polycationic Bioactive Chitosan Nanoparticles Inactivate Bacterial Endotoxins Annie Shrestha, BDS, MSc, PhD,* Martha Cordova, BSc, MSc,† and Anil Kishen, BDS, MDS, PhD* Abstract Introduction: The current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). Methods: Antimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a timedependent interaction with/without photoactivation (20 and 40 J/cm2). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide– and enzymelinked immunosorbent assay (tumor necrosis factor a and interleukin-6 expression)–based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. Results: CSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P < .005) even at 30 minutes of exposure. CSRBnps and MB with/without photoactivation significantly inactivated LPSs with reduced nitric oxide and tumor necrosis factor a expression (P < .05). Cell death and detachment after Ca(OH)2 treatment resulted in complete absence of all 3 inflammatory markers. Conclusions: Photodynamically activated CSRBnps caused significant inactivation of endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins. (J Endod 2015;-:1–6)
Key Words Chitosan, endotoxins, inactivation, nanoparticles, photodynamic
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n effective endodontic disinfection necessitates the elimination of bacterial biofilms and the inactivation of their by-products to facilitate optimal periradicular tissue healing (1). A chemomechanical approach that combines mechanical instrumentation and chemical irrigation has been shown to be reasonably effective in lowering viable bacterial loads in root canals (2, 3). Nevertheless, conventional protocols fall short in the elimination/inactivation of bacterial modulins such as endotoxins/ lipopolysaccharides (LPSs) from infected root dentin (4). LPSs have been associated with endodontic infections with aggravated clinical symptoms (5, 6). The continuous leakage of LPSs from the infected root canal system and its accumulation in the surrounding periradicular tissues have been shown in an animal model (6). Current topical antibacterials in endodontics such as 2.5% sodium hypochlorite and 2% chlorhexidine gel produced only an incomplete elimination of LPSs in vivo (4). The inability of the current topical disinfectants and medications to neutralize LPSs may be associated with the persistence of apical periodontitis in an endodontically treated tooth (4, 7). Furthermore, the existence of LPSs within root dentin and dentinal tubules (7) may negatively impact the possibility of biologically based treatment procedures in endodontics. A newer antimicrobial is essential that can overcome the current limitations of eliminating both bacteria and endotoxins from infected root dentin. LPSs are released from the cell wall of gram-negative bacteria, the predominant species involved in primary endodontic infections (5, 8). They possess a strong negative charge because of their lipid moiety, which could be neutralized and sequestered with positively charged molecules (9). Calcium hydroxide (Ca[OH]2) relies on the sustained establishment of a high pH in the root canal system. It requires a longer treatment period for the effective inactivation of LPSs (10). Although in vitro studies have supported the use of Ca(OH)2 in neutralizing LPSs (11, 12), an in vivo study conducted by Vianna et al (13) reported that LPSs remained in the root canal system even after the application of Ca(OH)2 dressing for 7 days. It is suggested that the clinical effectiveness of Ca(OH)2 may be hindered by the lack of hydroxyl ion delivery through dentinal tubules, complex canal anatomy, and the buffering capacity of dentin (10). Recently, photodynamic therapy (PDT), which applies a low-energy light with a photosensitizer, has been shown to have the potential to enhance the disinfection efficacy of the conventional chemomechanical preparation (14, 15). PDT is also effective in inactivating the virulence factors from gram-negative bacteria and reducing the inflammatory potential of LPSs adherent to a titanium implant (16, 17). Cationic nanoparticle–conjugated photosensitizers have been shown to potentiate the antibacterial efficacy of PDT (15, 18, 19). Bioactive nanoparticles of chitosan and rose bengal–conjugated nanoparticles (CSRBnps) have shown significant antibiofilm activity against both single-species and multispecies bacterial biofilms, even in the
From the *Discipline of Endodontics and †Dental Research Institute, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Anil Kishen, Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.12.007
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Basic Research—Technology presence of inhibitors such as dentin powder and organic remnants (15, 20, 21). Chitosan particles and their modifications have also been shown to be effective in neutralizing LPSs (22, 23). Combining the binding affinity of polycationic chitosan nanoparticles and the effect of singlet oxygen release by photoactivated photosensitizers, we hypothesized that CSRBnps will effectively neutralize bacterial LPSs, thereby reducing the expression of inflammatory cytokines from macrophage cells. Thus, the aim of this study was to evaluate the ability of photoactivated CSRBnps in the inactivation of bacterial endotoxins and direct toxicity to macrophage cells.
Materials and Methods Ultrapure LPSs from Pseudomonas aeruginosa (Sigma-Aldrich, St Louis, MO) (endotoxin level 500,000 EU/mg) were used in the study; 1 mg/mL LPSs was prepared as a stock solution. Low-molecular-weight, low-viscosity chitosan particles (Sigma-Aldrich) were used for the synthesis of chitosan nanoparticles (CSnps) (24) and CSRBnps following previous literature (15). All other chemicals were pyrogen free with the highest analytic grade unless specified. CSnps (10 mg/mL), Ca(OH)2 (10 mg/mL), CSRBnps (0.3 mg/mL), and methylene blue (MB) (10 mmol/L) were prepared fresh in culture medium. The noncoherent light source (Lumacare-LC-122M; LumaCare, Newport Beach, CA) with interchangeable fiber bundles and 30-nm band-pass filters at ranges 660 15 nm and 540 15 nm for MB and CSRBnps, respectively, was used.
Cell Culture RAW 264.7 macrophage cells at passages 3 to 5 (1 106 cells/ml) were used in the experiment. Cells were added into each well of a sterile 24-well plate (BD Falcon, Franklin Lakes, NJ) and incubated for 24 hours (37 C, 5% CO2) to allow cell attachment. Nonadherent cells were removed, and the remaining cells were washed with medium. Neutralization Treatment of LPS LPSs (500 EU/mL) were subjected to the following treatment groups: Ca(OH)2, CSnps, MB, and CSRBnps. The time-dependent effect on treatment with Ca(OH)2 and CSnps was evaluated at 6 and 12 hours (37 C) (22). Both time-dependent (15 minutes and 6 hours) and photoactivated (20 and 40 J/cm2, time: 4 and 8 minutes, respectively) effects were evaluated for MB and CSRBnps at the 2 incubation periods. In the negative control group, the culture medium was without LPSs (LPS ), and in the positive control group (LPS+), LPSs were added in culture medium, which was then added into the cells. The treated and untreated LPSs from different groups were then added into cells, and the plates were incubated (37 C, 5% CO2) for 12 hours. The cell-free supernatants were collected by centrifugation at 10,000 rpm for 5 minutes (4oC) and stored at 20 C for further experiments. Measurement of Nitric Oxide The levels of nitric oxide (NO) in the cell culture supernatants were quantified by measuring the nitrite using the Griess Reaction System (Promega, Madison, WI) according to the manufacturer’s instructions. The NO concentration was determined quantitatively using the standard curve of nitrite. Measurement of Tumor Necrosis Factor a and Interleukin 6 Tumor necrosis factor a (TNF-a) and interleukin (IL)-6 concentrations released by cells in culture medium were determined using an 2
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enzyme-linked immunosorbent assay with the Quantikine Human TNFa and IL-6 Immunoassay Kit (R&D Systems, Minneapolis, MN). Purified recombinant TNF-a and IL-6 were used as the standard. TNF-a and IL-6 were expressed as concentration per milliliter based on the standard curve.
Cell Viability Assessment The cells were assessed for viability following treatment with Ca(OH)2, CSnps, MB, and CSRBnps. Ca(OH)2 and CSnps for were measured for their time-dependent effect at 30 minutes and 12 hours (37 C). The effect of MB and CSRBnps on cells after photosensitization (15 minutes, 37 C) (dark toxicity) and photoactivation (20 and 40 J/ cm2) was evaluated. The control groups were tested without any treatment. Cell viability expressed as percentage survival was determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cells after treatment were examined under a light microscope to assess the direct effect of treatment on cells. The cells were washed after the treatment period and stained using 0.5% trypan blue for 5 minutes. The cells were viewed under a light microscope (Axio Vert.A1; Zeiss, Jena, Germany) at 20 magnification. Statistical Analysis All experiments were repeated 3 times in triplicate. The statistical significance was analyzed by 1-way analysis of variance and the Tukey post hoc test. The significance was set at 0.05.
Results NO Release The NO release by macrophage cells after treatment with or without LPSs was significantly different (Fig. 1A). The Ca(OH)2-treated groups showed a similar NO release to that of the LPS-negative group (11–12 mmol/L). CSnps showed a reduction of NO production compared with the LPS-positive group (P < .05). However, compared with the LPS-negative group, the NO release was twice as high in the CSnp groups (P < .005) at both time points. CSRBnps without photoactivation (0 J/cm2) showed a time-dependent neutralization effect that reduced the NO release significantly at 6 hours when compared with 15 minutes of treatment with LPSs (P < .05) (Fig. 1B). At both time points, the photoactivation resulted in significantly reduced NO release, which was comparable with the LPS-negative group. The MB group at both time points showed significantly reduced NO release from the cells (P < .05). Photoactivation of MB did not produce further inactivation of LPSs. TNF-a and IL-6 Release TNF-a and IL-6 release was completely absent in the absence of LPSs. Ca(OH)2 showed no TNF-a and IL-6 release from cells. The CSnp group showed a decrease in TNF-a release without any significant difference from the LPS-positive group (Fig. 2A). However, CSnps after 12 hours of interaction with LPSs significantly reduced IL-6 release (P < .05) (Fig. 2C). Both the CSRBnp and MB groups showed significantly better inhibition of TNF-a release at 15 minutes (P < .05) compared with 6 hours of interaction with and without photoactivation (Fig. 2B and D). At 6 hours, both the CSRBnp and MB groups showed TNF-a reduction only after photoactivation. IL-6 release was reduced significantly (P < .05) with CSRBnps at both time points without photoactivation. Photoactivation (40 J/cm2) of CSRBnps resulted in a highly significant (P < .005) reduction of IL-6 expression by the macrophage cells. The treatment with MB did not produce any significant reduction of IL-6 release. JOE — Volume -, Number -, - 2015
Basic Research—Technology Cell Viability Figures 1C and D and 3 show the effect of various treatment groups on the macrophage cells using a mitochondrial activity assay and light microscopy. CSnps were found to be nontoxic with 100% cell viability for up to 12 hours of incubation (Fig. 1C). Ca(OH)2 was highly cytotoxic to macrophage cells at the given concentration, which resulted in 70% cell death within 30 minutes. CSRBnps with or without photoactivation did not exhibit any toxicity. Although photoactivation with MB resulted in the reduction of cell survival, this change was not statistically significant (P = .23). The light microscopic images showed few dead cells attached on the plates of the Ca(OH)2 group (Fig. 3A). The trypan blue exclusion assay confirmed the presence of live and well-attached cells in all other treatment groups (Fig. 3B–D).
Discussion Gram-negative, aerobic bacteria such as Pseudomonas species have been associated with chronic infections and have been detected in persistent root canal infections (25, 26). The influence of LPSs from P. aeruginosa on macrophage cells was found to be significantly higher compared with that of Porphyromonas gingivalis in our preliminary experiments (data not shown). Endotoxins present in the root canals as well as within dentinal tubules could be contributed to persistent endodontic infection because they may act as a reservoir of continuous irritation to the periapical tissues (1, 4, 27). The high endotoxin concentration of 500 EU/mL used in this study correlated with endotoxins found in symptomatic root canal infections with pulp necrosis and apical periodontitis (28). Intracanal causes of the host immune response during periapical inflammation include the immune cells, intracellular mediators, effector molecules, and humoral antibodies (29). The presence of macrophages in human inflammatory periapical lesions has been long recognized. Neutrophils are known to dominate during the acute phase of the disease, and lymphocytes, macrophages, and plasma cells accumulate in the chronic phase of the disease (29). In chronic infection, macrophages are the primary innate immune cells to respond to invading microorganism. They play an important role in recognizing the molecular patterns on bacterial cells, initiating internalization and killing. Chronic infections are manifested by a cascade of immune reactions involving the generation of several cytokines, such as TNF-a, IL-1, IL-6, IL-8, effector molecules (eg, matrix metalloproteinase), NO, reactive oxygen species, and antibodies (30). TNF-a and IL-6 are considered to be the 2 major cytokines produced by the activated macrophages (29). The current study showed that PDT using CSRBnps and MB were effective in neutralizing LPSs, subsequently reducing the inflammatory potential of LPSs. Ca(OH)2 produced high cytotoxicity upon direct contact with the macrophage cells. The detachment and removal of the cells from plates resulted in the absence of any NO, TNF-a, and IL-6 production in the Ca(OH)2 group. The stand-alone effect of CSnps was not sufficient to reduce the LPS-induced inflammatory potential (NO and TNF-a) significantly. They only showed a reduction of IL-6 after 12 hours of interaction. Figure 1. NO release by macrophage cells stimulated with LPSs and treated with (A) Ca(OH)2 and CSnps and (B) CSRBnp and MB treatment with and without photoactivation. LPSs were allowed to react with CSRBnps and MB for 15 minutes and 6 hours before photoactivation. NO release in the LPS+ group was 27 mmol/L, and in the LPS group, it was 12 mmol/L. A cytotoxicity assay to assess the effect of (C) Ca(OH)2 and CSnps in a timedependent manner and (D) CSRBnps and MB after photoactivation of macrophage cells in vitro.
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Basic Research—Technology MB with/without photoactivation resulted in a slight decrease in cell viability, yet it produced a significant reduction of NO and TNF-a. However, no effect was observed on IL-6 production from activated macrophage cells. MB has been shown to form stable metachromatic complexes with LPSs of P. aeruginosa and Escherichia coli (31). The formation of MB dimers in the presence of LPSs could have aggregated and made LPSs unavailable to induce inflammatory mediators from macrophage cells. CSRBnps significantly neutralized LPSs after photoactivation. NO and IL-6 were reduced significantly even without photoactivation, whereas the inactivation of TNF-a required photoactivation. The effect of CSRBnps on reducing NO production without photoactivation was time dependent. This could be caused by the of LPS-CSRBnp complexes that start being formed in the initial phase of interaction (15 minutes) and are more stable after 6 hours of interaction. At 15 minutes, there could be LPSs that are either neutralized or still active, which result in variance in the production of NO from macrophages (Fig. 1B). The production of NO, IL-6, and TNF-a by macrophages has been used to determine the inflammatory potential of LPSs (12, 32). The neutralization of LPSs with CSRBnps and MB resulted in a decreased production of inflammatory cytokines in the present study. Marton and Kiss (32) reported an altered balance in the production and elimination of reactive oxygen intermediates in chronic apical periodontitis. TNF-a has a direct cytotoxic effect and a general debilitating effect in chronic disease. Macrophage-derived TNF-a has numerous systemic and local effects (30). The presence of TNF-a has been reported in human apical periodontitis lesions and in the root canal exudates of teeth with apical periodontitis. NO is synthesized by a complex family of enzymes called NO synthases. NO is reactive nitrogen intermediate and one of the initial and nonspecific responses to infection by macrophages. NO synthesis is increased in periapical infection as a result of macrophage infiltration in the periapical tissues (33). Both CSRBnps and MB were more effective in reducing NO expression in the present study. NO plays an important role in the pathogenesis of periapical lesions either by directly or indirectly modulating the production of other proinflammatory cytokines such as IL-6 and TNF-a. These cytokines are highly potent inflammatory markers, and only CSRBnps were able to significantly inhibit IL-6 and TNF-a expression. However, the present study is not able to explain the higher efficacy of CSRBnps without photoactivation to reduce IL-6 expression compared with TNF-a. Studies to understand the effect of the LPS structure on the expression of specific cytokines are needed to explain this observation. Chitosan is a polycation that possesses a positive charge in each of its glucosamine units (34). They formed stable complexes with negatively charged bacterial endotoxins, thereby reducing the inflammatory potential of activated macrophages (23). However, care should be taken to use chitosan of low molecular weight to avoid gelation caused by charge interaction. In this study, we used a lowmolecular-weight, low-viscosity chitosan to synthesize nanoparticles and photosensitizer-conjugated nanoparticles (20). CSnps alone were not as effective as CSRBnps toward LPS neutralization. The LPS neutralization effect of CSRBnps may be attributed to the smaller size of CSRBnps (60 nm) compared with CSnps (80 nm) (ie, a positive
Figure 2. (A and B) TNF-a and (C and D) IL-6 secretion by macrophage cells stimulated with LPSs and treated with Ca(OH)2, CSnps, CSRBnps, and MB. The interaction of LPSs with Ca(OH)2 and CSnps was assessed with time, and CSRBnps and MB were assessed with and without photoactivation. TNF-a expression in the LPS+ group was 5.14 ng/mL, and in the LPS group, it was 0.02 ng/mL. IL-6 expression in the LPS+ group was 795 pg/mL, and in the LPS group, it was 0.09 pg/mL.
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Figure 3. Light microscopic images of macrophage cells after direct contact with (A) Ca(OH)2, (B) CSnps, (C) CSRBnps, and (D) MB. The cells were stained with trypan blue stain to assess the cell morphology and membrane damage after the initial 30 minutes of treatment. The black arrows show dead cells or disrupted cell mass with trypan blue uptake.
charge [15] with a high affinity toward LPSs). The singlet oxygen produced upon photoactivation might further disintegrate the lipid moiety of LPSs. The PDT parameters were chosen based on previous studies that resulted in complete elimination of bacterial biofilms in root canal models (15, 18). Burns et al (9) have reported that small molecules with 2 protonable cationic groups effectively bind and neutralize endotoxins by interacting with the phosphate moieties of the lipid A component (9). PDT has shown promising results in eliminating endodontic bacteria/biofilms (14, 15), yet investigations examining its effect on LPSs are relatively limited (17). In the current study, photoactivation of CSRBnps resulted in significant neutralization of endotoxins even at a high concentration. CSRBnps have been previously shown to exhibit significant antibiofilm efficacy (15), stabilize dentin collagen after photo– cross-linking, and inhibit collagenolytic activity (35). Furthermore, the importance of selecting agents that could selectively inactivate LPSs while sparing the mammalian cells is highly beneficial in a clinical scenario in which treatment of infected tissue is in close proximity to normal host tissues (intraradicular and extraradicular regions close to the apical foramen). Considering these findings, CSRBnps could serve as an efficient antimicrobial agent for endodontic application.
Acknowledgments Supported by the American Association of Endodontists Foundation, Research Grant Program (Fund #494223), University of Toronto (Fund #928133), and Canadian Foundation of Innovation (Fund #493891). The authors deny any conflicts of interest related to this study.
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