1 Photoactivated rose bengal functionalized chitosan nanoparticles produce 2 antibacterial/biofilm activity and stabilize dentin-collagen 3 Annie Q1 Shrestha, MSc a , Michael R. Hamblin, PhD b,c,d , Anil Kishen, PhD a, ⁎ 4 a Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada 5 b Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA 6 c Department of Dermatology, Harvard Medical School, Boston, MA, USA 7 d Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA 8 Received 2 July 2013; accepted 28 October 2013 9 Abstract 10 Treatment of infected teeth presents two major challenges: persistence of the bacterial-biofilm within root canals after treatment and 11 compromised structural integrity of the dentin hard-tissue. In this study bioactive polymeric chitosan nanoparticles functionalized with rose- 12 bengal, CSRBnp were developed to produce antibiofilm effects as well as stabilize structural-integrity by photocrosslinking dentin-collagen. 13 CSRBnp were less toxic to fibroblasts and had significant antibacterial activity even in the presence of bovine serum albumin. CSRBnp 14 exerted antibacterial mechanism by adhering to bacterial cell surface, permeabilizing the membrane and lysing the cells subsequent to 15 photodynamic treatment. Photoactivated CSRBnp resulted in reduced viability of Enterococcus faecalis biofilms and disruption of biofilm 16 structure. Incorporation of CSRBnp and photocrosslinking significantly improved resistance to degradation and mechanical strength of 17 dentin-collagen (P b 0.05). The functionalized chitosan nanoparticles provided a single-step treatment of infected root dentin by combining 18 the properties of chitosan and that of photosensitizer to eliminate bacterial-biofilms and stabilize dentin-matrix. 19 © 2013 Published by Elsevier Inc. 20 Key words: Functionalized nanoparticles; Chitosan; Biofilms; Collagen; Dentin; Photodynamic therapy 21 22 Q2 Introduction 23 Approximately 60% of human infections are associated with 24 bacterial-biofilms, which include both implant-related infections 25 and chronic non-implant related infections. 1 Likewise the rate of 26 root canal treatment failure of infected teeth has not decreased 27 below 18-26% even with advanced therapeutic options to 28 improve the treatment outcome. 2,3 The widespread recognition 29 of biofilm as the main factor in dental infection has led research 30 towards improved antimicrobial treatment strategies. 4 Another 31 significant issue in antimicrobial treatments is that the agents 32 need to be selective in eliminating bacteria while sparing the 33 adjacent mammalian cells, which allows targeted antibacterial 34 activity. Targeted antibacterial activity is an advantageous 35 feature in infected hard tissue management. Compromised 36 mechanical integrity and chemical stability due to disease- 37 mediated degradation of the dentin hard tissues caused by host/ 38 bacterial proteases 5 , and treatment-associated tissue changes 39 have been reported. 6 However, little attention has been paid 40 towards improving the chemical stability and mechanical 41 properties of these previously infected hard tissues. Thus the 42 two major challenges in the management of infected dental hard 43 tissue are (1) decontamination of bacterial-biofilm from the root 44 canals and (2) repair of disease-mediated hard tissue changes. 45 Although approaches that counter these challenges should lead 46 to improved treatment outcomes, currently there is no treatment 47 in dentistry that would produce significant antibiofilm efficacy 48 and at the same time enhance the ultrastructural integrity of 49 dentin tissue in infected teeth. 50 PDT has been applied in biomedicine owing to its broad- 51 spectrum antimicrobial activity 7 and ability to produce cross- 52 linking of proteins and collagen. 8,9 The singlet oxygen produced 53 facilitates the formation of inter- and intra-molecular covalent This work is funded by the University of Toronto and CIHR Training Fellowship (TGF-53877). MRH was funded by the US National Institutes of Health (R01AI50875). ⁎ Corresponding author. Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, ON M5G 1G6, Canada. E-mail address: anil.kishen@utoronto.ca (A. Kishen). Nanomedicine: Nanotechnology, Biology, and Medicine xx (2013) xxx – xxx nanomedjournal.com 1549-9634/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.nano.2013.10.010 NANO-00847; No of Pages 11 Please cite this article as: Shrestha A., et al., Photoactivated rose bengal functionalized chitosan nanoparticles produce antibacterial/biofilm activity and stabilize dentin-collagen. Nanomedicine: NBM 2013;xx:1-11, http://dx.doi.org/10.1016/j.nano.2013.10.010