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pH-activated Nanoparticles for Controlled Topical Delivery of Farnesol to Disrupt Oral Biofilm Virulence Benjamin Horev, Marlise I Klein, Geelsu Hwang, Yong Li, Dongyeop Kim, Hyun Koo, and Danielle S. W. Benoit ACS Nano, Just Accepted Manuscript • DOI: 10.1021/nn507170s • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 12, 2015
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pH-activated Nanoparticles for Controlled
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Topical Delivery of Farnesol to Disrupt Oral
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Biofilm Virulence
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Benjamin Horev,1,§ Marlise I. Klein,2,‡,§, Geelsu Hwang, 3 Yong Li, 3,∞ Dongyeop Kim, 3,∞
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Hyun Koo,2,3,4,*,∞, and Danielle S.W. Benoit1,5,6 1
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Department of Biomedical Engineering, University of Rochester, NY 14627, United States
Center for Oral Biology, University of Rochester, NY 14627, United States 3Biofilm Research Lab, Levy Center for Oral Health, University of Pennsylvania, PA 19104, United States,
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Department of Orthodontics and Divisions of Pediatric Dentistry and Community Oral Health,
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School of Dental Medicine, University of Pennsylvania, PA 19104, United States, 5Department
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of Chemical Engineering, University of Rochester, NY 14627, United States, 6Center of
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Musculoskeletal Research, University of Rochester, NY 14627, United States, ‡Current address:
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Department of Dental Materials and Prosthodontics, Araraquara Dental School, Univ Estadual
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Paulista, UNESP, Sao Paulo, Brazil.
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* Address correspondence to:
[email protected];
[email protected] 16
§
17
∞
Authors contributed equally to this work.
Were involved in in vivo efficacy testing herein.
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ACS Nano
Abstract
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Development of effective therapies to control oral biofilms is challenging, as topically
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introduced agents must avoid rapid clearance from biofilm-tooth interfaces while targeting
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biofilm microenvironments. Additionally, acidification of biofilm microenvironments is
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associated with cariogenic (caries-producing) biofilm virulence. Thus, nanoparticle carriers
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capable of binding to hydroxyapatite (HA), saliva-coated HA (sHA), and exopolysaccharides
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with enhanced drug-release at acidic pH were developed. Nanoparticles are formed from diblock
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copolymers composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA), butyl methacrylate
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(BMA), and 2-propylacrylic acid (PAA) (p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PAA)) that
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self-assemble into ~21 nm cationic nanoparticles. Nanoparticles exhibit outstanding adsorption
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affinities (~217 L-mmol-1) to negatively-charged HA, sHA, and exopolysaccharide-coated sHA
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due to strong electrostatic interactions via multivalent tertiary amines of p(DMAEMA). Owing
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to hydrophobic cores, Nanoparticles load farnesol, a hydrophobic antibacterial drug, at ~22 wt%.
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Farnesol release is pH-dependent with t1/2=7 and 15 h for release at pH 4.5 and 7.2, as
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Nanoparticles undergo core destabilization at acidic pH, characteristic of cariogenic biofilm
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microenvironments. Importantly, topical applications of farnesol-loaded nanoparticles disrupted
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Streptococcus mutans biofilms 4-fold more effectively than free farnesol. Mechanical stability of
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biofilms treated with drug-loaded nanoparticles was compromised, resulting in >2-fold
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enhancement in biofilm removal under shear stress compared to free farnesol and controls.
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Farnesol-loaded nanoparticles effectively attenuated biofilm virulence in vivo using a clinically-
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relevant topical treatment regimen (2x/day) in a rodent dental caries disease model. Treatment
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with farnesol-loaded nanoparticles reduced both the number and severity of carious lesions,
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while free-farnesol had no effect. Nanoparticles have great potential to enhance the efficacy of
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antibiofilm agents through multi-targeted binding and pH-responsive drug release due to
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microenvironmental triggers.
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Keywords:
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exopolysaccharides, matrix, dental caries, Streptococcus mutans biofilms, farnesol
polymeric
micelles,
pH-responsive,
nanoparticles,
dental
pellicle,
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The majority of persistent infectious diseases in humans are caused by virulent biofilms,
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including those occurring in the mouth, e.g., dental caries and periodontal disease.1, 2 The annual
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treatment cost of oral biofilm-related diseases is ~$81 billion in the US alone.3 Biofilms develop
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when microbes accumulate on surfaces, forming structured communities encapsulated within an
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extracellular matrix comprised of polymeric substances such as exopolysaccharides (EPS).2, 4, 5
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In the oral cavity, caries-producing (cariogenic) biofilms assemble on pellicle-covered teeth, as
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EPS-rich matrix rapidly develops in the presence of dietary sucrose.6-9 EPS, produced by
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bacterial exoenzymes (e.g., glucosyltransferases), promote local accumulation of pathogens (e.g.,
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Streptococcus mutans) while forming a diffusion-limiting polymeric matrix that protects the
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embedded bacteria.2,
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creating highly acidic microenvironments.7, 8, 10 In human dental biofilm, also known as plaque,
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pH values often reach pH~4.5 or even lower, particularly after exposure to sucrose, starch, and
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other cariogenic food products.11-14 At sites of active caries, persistent acidic plaque of pH~4.5-
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5.5 can result.11-14 The low pH niches induce EPS synthesis while S. mutans and other cariogenic
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organisms thrive,15 ensuring continuous biofilm accumulation, acid-dissolution of tooth enamel,
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and ultimately, the onset of carious lesions.7-10, 16 Resident microorganisms become recalcitrant
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to antimicrobial therapies, stemming from a combination of bacterial drug resistance and reduced
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drug bioavailability and persistence within biofilm microenvironments.1, 2
4, 5
In parallel, sugars are fermented by bacteria within the EPS matrix,
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ACS Nano
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Development of novel therapeutic approaches against oral biofilms is challenging, as topically
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introduced antibacterial agents are not retained at necessary concentrations for prolonged periods
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due to rapid clearance by saliva. There exists a need to enhance the bioavailability and retention
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of antibacterial agents at the dental surfaces and within the biofilm. Fundamental understanding
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of biofilm matrix assembly and changes in the biofilm microenvironment offers opportunities to
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exploit drug delivery systems. Several strategies have been developed. These include materials
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with inherent antimicrobial properties such as cationic liposomes and silver particles,2,
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well as drug delivery systems with specific affinity to tooth surfaces.19,
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approaches are mostly designed to target hydroxyapatite, the mineral component of tooth
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enamel, rather than the pellicle, the proteinaceous film that covers hydroxyapatite-based enamel
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in the mouth, or EPS. None of these strategies exploits the acidic milieus within EPS-rich
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biofilm microenvironments to trigger drug delivery. Nevertheless, delivery systems have been
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developed to release drugs or biologically-active molecules in response to other acidic
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environments, such as in tumors, cellular endosomes or lysosomes, or sites of bacterial
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infections.20, 26-29 These include acid-degradable residues 30, 31 and nanoparticles that incorporate
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pH-responsive
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dimethylaminoethyl methacrylate (DMAEMA),34,
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combinations thereof26-29, 39 to trigger drug delivery via nanoparticle destabilization at low pH.
residues
such
as
diethylaminoethyl 35
methacrylate
21-25
17-20
as
However, these
(DEAEMA),32,
33
propylacrylic acid (PAA),36-38 or
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There exists an opportunity to advance drug delivery approaches for oral biofilms by providing
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localized drug release at the pellicle/biofilm interface and within EPS-rich matrix in response to
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acidic pH niches, where pathogenic (cariogenic) bacteria prosper and actively develop biofilms.
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To address this challenge, nanoparticles with exciting properties for antibiofilm delivery were
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developed. We describe a pH-activated polymer-based nanoparticles that bind with high affinity
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to tooth surfaces (hydroxyapatite and pellicle) and EPS, enhancing drug retention at at-risk sites
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for biofilm development. The nanoparticles contain pH-responsive moieties that expedite drug
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release at acidic pH values found within cariogenic biofilm microenvironments, resulting in
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excellent antibiofilm activity and effectively reducing the onset of carious lesions in vivo.
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Specifically, nanoparticles composed of cationic poly(dimethylaminoethyl methacrylate)
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(p(DMAEMA)) coronas and hydrophobic and pH-responsive p(DMAEMA-co-BMA-co-PAA)
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cores30,36 were used to achieve high affinity, multivalent binding to tooth surfaces and EPS. A
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hydrophobic antibacterial agent, farnesol, effective against planktonic S. mutans cells, but with
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limited activity against cariogenic biofilms following topical applications,43 was used to
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demonstrate enhanced drug efficacy through high capacity nanoparticles-mediated delivery.
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Altogether, we show that the developed nanoparticle composition and formulation exhibits
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outstanding binding affinities to pellicle and EPS surfaces (~217 L-mmol-1) as well as drug
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loading capacities (up to ~22 wt%), and pH-dependent drug release. Farnesol was released
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rapidly at acidic pH due to protonation of DMAEMA and PAA residues within nanoparticle
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cores and resulting destabilization of nanoparticles structure. Topical applications of farnesol-
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loaded nanoparticles that target and localize high farnesol depots on surfaces and within EPS
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matrix, disrupted S. mutans biofilms 4-fold more effectively than free farnesol. The mechanical
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stability of treated biofilms was compromised, resulting in >2-fold enhancement in biofilm
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removal from pellicle-coated apatitic surfaces upon exposure to shear stress, compared to free
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farnesol treatments or to controls. Ultimately, the efficacy of nanoparticle-mediated drug
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delivery on biofilm virulence was demonstrated through reductions in both the incidence and the
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severity of carious lesions in vivo, using a clinically relevant twice-daily topical treatment
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regimen. The achieved drug retention and subsequent in vivo therapeutic effect of topically
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applied nanoparticles is a highly desirable property for development of novel and efficacious
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therapies for biofilm-related oral diseases such as dental caries.6
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Results and Discussion
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Polymer structure and function
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All polymers used in this work were synthesized via reversible addition-fragmentation chain
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transfer (RAFT) polymerizations, which provides precise control over polymer molecular
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weights and polydispersity indices (Mw/Mn, PDI0.98). Initial release rate (B. inset, r0), release rate constant (kobs) and half-time of release (t1/2) at pH 4.5 suggest 2-fold faster release at pH 4.5 as compared to pH pH 7.2. D. Antibacterial activity of farnesol-loaded nanoparticles at pH 7.2. A ~2.4 log decrease in bacterial viability was observed after 1 h of exposure to drug-loaded nanoparticles. Error bars represent standard deviation (n=3 independent experiments) for drug release experiments, and standard error (n=7) for antibacterial activity experiments. Asterisks denote significant differences at p
