antibacterial nanoparticles in endodontics: a review · antibacterial nanoparticles in endodontics:...

Review Article

Antibacterial Nanoparticles in Endodontics:A Review
Annie Shrestha, BDS, MSc, PhD, and Anil Kishen, BDS, MDS, PhD
Abstract
Significance
Nanoparticles with their enhanced and uniquephysicochemical properties hold new prospectsfor the treatment and prevention of dental infec-tions. The near future potential of nanoparticles inclinical endodontics warrants sound researchbased on scientific and clinical collaborations.

Introduction: A major challenge in root canal treat-ment is the inability of the current cleaning and shapingprocedures to eliminate bacterial biofilms survivingwithin the anatomic complexities and uninstrumentedportions of the root canal system. Methods: Nanopar-ticles with their enhanced and unique physicochemicalproperties, such as ultrasmall sizes, large surface area/mass ratio, and increased chemical reactivity, have ledresearch toward new prospects of treating and prevent-ing dental infections. This article presents a comprehen-sive review on the scientific knowledge that is availableon the application of antibacterial nanoparticles in end-odontics. Results: The application of nanoparticles in theform of solutions for irrigation, medication, and as an ad-ditive within sealers/restorative materials has been eval-uated to primarily improve the antibiofilm efficacy in rootcanal and restorative treatments. In addition, antibiotic orphotosensitizer functionalized nanoparticles have beenproposed recently to provide more potent antibacterial ef-ficacy. Conclusions: The increasing interest in this fieldwarrants sound research based on scientific and clinicalcollaborations to emphasize the near future potential ofnanoparticles in clinical endodontics. (J Endod2016;42:1417–1426)

Key WordsAntibacterial, chitosan, functionalized, nanoparticles,silver

From the Discipline of Endodontics, University of Toronto,Toronto, Ontario, Canada.

Address requests for reprints to Prof Anil Kishen, Disciplineof Endodontics, Faculty of Dentistry, University of Toronto, 124Edward Street, Toronto, ON M5G 1G6, Canada. E-mail address:[email protected]/$ - see front matter

Copyright ª 2016 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2016.05.021

JOE — Volume 42, Number 10, October 2016

Nanomaterial denotesa natural, incidental,

or manufactured materialcontaining particles in anunbound state or as anaggregate or agglomeratein which 50% or more ofthe particles in number,size, distribution, or 1 or
more external dimensions is in the size range of 1–100 nm (1). Nanomaterials offerunique physicochemical properties, such as ultrasmall sizes, large surface area/massratio, and increased chemical reactivity, compared with their bulk counterparts(2, 3). The increased surface to volume ratio and increased number of atoms thatare present near the surface compared with micro-/macrostructures are suggestedto contribute to the distinctly different properties of nanomaterials. These advantagesmay be exploited to design highly specific materials and devices to interact with atthe subcellular and molecular level of the human body in order to achieve maximaltherapeutic efficacy with minimal side effects (4, 5).
Nanotechnology has progressed rapidly in science and technology, creating amyriad of biomedical applications such as drug delivery, tissue regeneration, antimicro-bial application, gene transfection, and imaging (2, 3, 5, 6). The term nanodentistryimplies the application of nanomaterials and dental nanorobots toward diagnosis andtreatment, with the goal of improving comprehensive oral health. The scope of suchstrategies includes a wide variety of oral health–related issues such as treatment ofdentin hypersensitivity, biofilm elimination, diagnosis and treatment of oral cancers,bone replacement materials, and so on. In the field of endodontics, the developmentof nanomaterials is focused on steps that would improve antimicrobial efficacy,mechanical integrity of previously diseased dentin matrix, and tissue regeneration.Currently, newer technologies are being tested in endodontics, mainly towardovercoming the microbial challenge (7, 8).

Based on the composition, nanoparticles are generally classified as either natu-rally occurring or synthetic (Table 1). They are further categorized as organic or inor-ganic in nature. Based on the shape, they are classified as particles, spheres, tubes, rods,plates, and so on. Functionalized nanoparticles are those that have a core of 1 materialand additional molecules or proteins bonded on its surface or encapsulated within.Depending on the specific applications, nanoparticles can be functionalized with pep-tides, drugs, photosensitizers, and so on (9, 10). The core nanoparticles can be usedas a convenient surface for molecular assembly and may be composed of inorganic ororganic materials. An additional layer of linker molecules is required to proceed withfunctionalization wherein the linker molecules have reactive groups at both ends thatbind various moieties like biocompatibles (dextran), antibodies, fluorophores, andso on onto the core nanoparticle.

This review aimed to provide comprehensive information on the scientific knowl-edge that is available for the use of nanotechnology toward antibacterial applications inendodontics.

Antibacterial NanoparticlesBacterial biofilms are considered the major cause of both primary and secondary

root canal infection (11, 12). Conventionally, chemical antimicrobials are used topically

Antibacterial Nanoparticles 1417

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TABLE 1. Nanoparticles Available Based on the Composition

Inorganic Metallic Polymeric Quantum dots Functionalized with

Zinc oxide Gold Alginate Cadmium sulfide DrugsIron oxide Silver Chitosan Cadmium selenide PhotosensitizersTitanium dioxide Iron AntibodiesCerium oxide Copper ProteinsAluminum oxide

bioactive glassMagnesium

Review Article

within the root canals in combination withmechanical instrumentation toachieve effective microbial reduction before filling the root canal with aninert filling material (7, 8, 13). The current level of evidence showed thatdespite the advancements in treatment strategies the rate of treatmentfailure has not decreased below 18%–26% for the past 4 to 5 decades(14–16). This could be mainly attributed to the inability or limitationsof current technologies to deal with the disease process as a whole(17). Other than this, the conservative management of infectionsinvolving topical or systemic antibiotics has been shown to be ineffectivebecause of several challenging factors (Table 2) (19, 20). Furthermore,the use of antibiotics is highly debatable, as ‘‘In the ongoing war againstantibiotics, the bacteria seem to be winning, and the drug pipeline isverging on empty’’ (21).

Because of the shortcomings of current antibiofilm strategies inroot canal treatment, advanced disinfection strategies are being devel-oped and tested. We review newer antibacterial nanoparticles that havebeen introduced at the laboratory levels with significant potential foreliminating endodontic biofilms.

Chitosan NanoparticlesChitosan (poly[1,4-b-D-glucopyranosamine]), a deacetylated de-

rivative of chitin, is the second most abundant natural biopolymer.Nanoparticles of chitosan could be synthesized or assembled usingdifferent methods depending on the end application or the physicalcharacteristics required in the nanoparticles (22). Chitosanhas received significant interest in biomedicine (22–24) because ofits versatility in various forms such as powder (micro- andnanoparticles), capsules, films, scaffolds, hydrogels, beads, andbandages (22). Chitosan has a structure similar to extracellular matrix

TABLE 2. Limitations of Current Topical or Systemic Antimicrobial TreatmentStrategies to Manage Infectious Diseases

Cause and treatment Challenges/limitations

Microbial factorsAntibiotic-resistant

mutant strainsMethicillin-resistant Staphylococcus

aureusVancomycin-resistant enterococci

Antibiotic-resistantmechanisms

Exchange of genetic materialsDeficiency of specific porin channelsPromotion of active drug effluxThickening of the peptidoglycan layer

of the outer wallStructure and

organizationof microbes

Variation in the outer wall in differentclasses of bacteria and fungi

Formation of biofilmsProtozoal existence as tropic feeding

stage of resting cystic stageAntibiotic misuse Excessive or inappropriate prescription

Failure to complete treatment regimenWidespread use of antibiotics in

livestock feedstuff

Data from Cunha BA. Antibiotic resistance. Control strategies. Crit Care Clin 1998;14:309–27 (18);

Finegold SM. Intestinal microbial changes and disease as a result of antimicrobial use. Pediatr Infect

Dis 1986;5:S88–90 (19); and Cookson BD. The emergence of mupirocin resistance: a challenge to

infection control and antibiotic prescribing practice. J Antimicrob Chemother 1998;41:11–8 (20).

1418 Shrestha and Kishen

components and is therefore used to reinforce the collagen constructs(25). This hydrophilic polymer with a large number of hydroxyl andfree amino groups can be subjected to numerous chemical modifica-tions and grafting, resulting in functionalization, which is discussedin a separate section (26–28). Nanoparticles of chitosan have beendeveloped mainly for antibacterial and drug/gene delivery applications.

Chitosan has excellent antibacterial, antiviral, and antifungal proper-ties (29). In case of bacteria, gram-positive bacteria weremore susceptiblethan gram-negative ones. The minimum inhibitory concentrations rangedfrom 18–5000 ppm depending on the organism, pH, degree of deacetyla-tion (DD), molecular weight, chemical modifications, and presence oflipids and proteins (29, 30). DD is known to influence the antibacterialactivity. With higher DD, the number of amine groups increases perglucosamine unit, and, thus, chitosan showed higher antibacterialefficacy (31). Chitosan nanoparticles (CS-NPs) by virtue of their chargeand size are expected to possess enhanced antibacterial activity.

Mechanism of ActionThe proposed mechanism of action is contact-mediated killing

that involves the electrostatic attraction of positively charged chitosanwith negatively charged bacterial cell membranes (Fig. 1). This mightlead to altered cell wall permeability, eventually resulting in the ruptureof cells and leakage of the proteinaceous and other intracellular com-ponents (29, 32). Under transmission electron microscopy, thebacterial cells were noted to be completely enveloped in the chitosan,forming an impermeable layer (33). This could result in the preventionof transport of essential solutes leading to cell death. In case of fungi,chitosan was hypothesized to enter the cell and reach the nucleus,bind with DNA, and inhibit RNA and protein synthesis.

Current ApplicationsFor the first time, Kishen et al (34) looked into the efficacy of

various cationic nanoparticles to improve root canal disinfection. Dentintreated with nanoparticles resulted in significantly reduced adherence ofEnterococcus faecalis. The antibacterial efficacy of CS-NPs and zinc ox-ide in disinfecting and disrupting E. faecalis (ATCC and OG1RF) biofilmswas evaluated later on (35). These nanoparticles eliminated biofilms ona concentration- and time-dependent manner and also retained theirantibacterial properties after aging for 90 days (35). CS-NPs can be deliv-ered within the anatomic complexities and dentinal tubules of an infectedroot canal to enhance root canal disinfection (36). Biofilm bacteria areknown to express efflux pumps as a resistance mechanism to antimicro-bials (37). When tested along with known efflux pump inhibitors, theantibacterial efficacy of CS-NPs was not affected against bacterial biofilmscompared with cationic photosensitizers (38). Another challenge in us-ing antibacterial agents inside the root canal space is the neutralizing ef-fect of different tissue inhibitors (39). Similarly, tissue inhibitors such aspulp and serum albumin inhibited the antibacterial effect of CS-NPssignificantly (40), whereas dentin, the dentin matrix, and lipopolysac-charides did not affect the efficacy of CS-NPs. In another in vitro study,CS-NPs were used in combination with different brands of chlorhexidineto eliminate E. faecalis with potential application toward tissue regener-ation using membrane barriers in periapical surgery (41). The addition


Figure 1. A schematic diagram showing the antibacterial mechanism of nanoparticles with positive charge (eg, chitosan). Mature bacterial biofilm consisting ofabundant extracellular polymeric substance (EPS) and bacteria enclosed. When cationic nanoparticles are introduced for the treatment of biofilms, it can interactwith both EPS and bacterial cells. The initial electrostatic interaction between positively charged nanoparticles and negatively charged bacterial surface. Bacterialkilling occurs upon contact-mediated lipid peroxidation via the production of ROS. The membrane damage and increased permeability of unstable membraneeventually lead to ingress of nanoparticles into the cytoplasm and release of cytoplasmic constituents. EPS secreted by bacteria in biofilm may interact with thenanoparticles and prevent them from interacting with bacteria, thus reducing antibacterial efficacy.

Review Article

of CS-NPs provided a significantly greater reduction of colony-formingunits in agar plates as well as infected collagen membranes.

Clinical SignificanceInitial studies highlighted that cationic CS-NPs hold significant

potential to achieve improved root canal disinfection. However, the pro-longed treatment time required to achieve effective bacterial eliminationand the effect of tissue inhibitors presented asmajor setbacks to CS-NPs.This warranted methods to overcome these shortcomings in futureresearch toward the application of CS-NPs.

Bioactive GlassBioactive glass (BAG) received considerable interest mainly

because of its osteoinductive effect and antibacterial properties towardvarious orthopedic and dental applications. The antibacterial activity of


BAGs has been investigated using 3 different approaches (Fig. 2A–C)(42–45). BAG consists of SiO2, Na2O, CaO2, and P2O5 at differentconcentrations and depends on the local physiological changes for itsantibacterial effects (45).

Mechanism of ActionThe antibacterial activity of BAG depends on the following factors

acting simultaneously (45):1. High pH: An increase in pH because of release of ions in an aqueous

environment2. Osmotic effects: An increase in osmotic pressure above 1% is inhib-

itory for many bacteria3. Ca/P precipitation: Induces mineralization on the bacterial surface

Furthermore, the release of Ca 2+, Na+, PO43�, and Si4+ couldlead to the formation of bonds with the mineralized hard tissues.


Figure 2. (A) One approach has focused on specially formulated BAGs that can dramatically change the local physiological conditions when they areimplanted to produce a bactericidal effect. (B) Another approach is to dope the BAG during its manufacture with trace quantities of elements (eg, Ag)that are known for their antibacterial activity, and, as the glass degrades, those elements are released at a clinically desirable rate. (C) The thirdapproach is to use BAG in conjunction with antibiotics. (Reprinted from Materials Science and Engineering C, volume 41, Rahaman MN, Bal BS,Huang W, Review: emerging developments in the use of bioactive glasses for treating infected prosthetic joints, pages 224–31, Copyright 2014, withpermission from Elsevier.)

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Current ApplicationsBAGs in micro- and nanoforms have been tested to improve root

canal disinfection (46–48). The nanometric BAG used by Zehnder et al(47) was amorphous in nature, ranging from20–60 nm in size. In vitroroot canal disinfection studies showed a significantly less antibacterialeffect of BAG compared with calcium hydroxide in preventing residualbacterial growth (47). Waltimo et al (44) suggested that an ideal prep-aration of 45S5 BAG suspensions/slurries for root canal disinfectionshould combine a high pH induction with a capacity for continuingrelease of alkaline species. Despite the higher specific surface area ofnanometric BAG, the micrometric counterpart had a considerablyhigher alkaline capacity and eliminated biofilms significantly better(44). Planktonic bacteria were killed significantly better comparedwith biofilm bacteria (49, 50). The reduced antibacterial efficacy ofnano-BAG compared with micro-BAG has been mainly contributed tothe 10-fold increase in silica release and solution pH elevation bymore than 3 units by the latter (49).

Clinical SignificanceThe conflicting evidence on BAG microparticles and nanoparticles

to generate a significant antibacterial effect warrants further researchbefore clinical application.


Silver NanoparticlesSilver compounds and nanoparticles have been widely used in

biomedicine, mainly because of their antibacterial property (51, 52).In case of dental application, silver and its nanoparticles have beentested for application as dental restorative material, endodonticretrograde filling material, dental implants, and caries inhibitorysolution (53).

Mechanism of ActionSilver is known to produce an antibacterial effect by acting on mul-

tiple targets starting from interaction with the sulfhydryl groups of pro-teins and DNA, alter the hydrogen bonding/respiratory chain, unwindDNA, and interfere with cell-wall synthesis/cell division (54, 55).Silver nanoparticles (Ag-NPs) are known to further destabilize thebacterial membrane and increase permeability, leading to leakage ofcell constituents (51, 56).

Current ApplicationsHiraishi et al (57) tested silver diamine fluoride against E. fae-

calis biofilms in vitro and showed complete elimination of 48-hourbiofilms after 60 minutes of interaction; 3.8% silver diamine fluoridewas also found deposited on the surface of dentin and penetrated up


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to 40mm into dentinal tubules. Another study showed that the antibio-film efficacy of Ag-NPs was significant when applied as a medicamentand not as an irrigant (58); 0.02% Ag-NP gel as medicament for 7 dayswas found to be significantly better in E. faecalis biofilm disruptioncompared with calcium hydroxide groups and syringe irrigationwith higher concentration Ag-NP (0.1%) solution. They suggestedthat when used as medicament there is a prolonged interaction be-tween positively charged Ag-NPs and negatively charged biofilm bac-teria/structure resulting in this difference. Ag-NP suspension whencombined with calcium hydroxide showed significantly reduced E.faecalis from root canal dentin (59). Another recent study showedvariation of antibacterial efficacy of Ag-NPs with charge and in thepresence of dentin as a tissue inhibitor (60). Ag-NPs of 9 nm sizeand positive charge had the smallest minimal inhibitory concentrationagainst planktonic E. faecalis when compared with sodium hypochlo-rite, chlorhexidine, and the other tested Ag-NPs. Positively chargedAg-NPs completely prevented the growth of planktonic E. faecalisin as short as 5 minutes of contact time and retained the antibacterialefficacy even in the presence of dentin. In addition, these Ag-NPs werefound to be cytocompatible to fibroblast cells. Ag-NP–loaded meso-porous bioactive glass (Ag-MBG) was tested against 4-week-old E.faecalis biofilms in root canals. A significant disruption of the biofilmstructure was observed compared with mesoporous bioactive glassand calcium hydroxide, which has been mainly contributed to sus-tained Ag ions released from the mesoporous structure (42). All thesepositive results with in vitro studies warrant further experiments inclinically relevant in vitro models/in vivo situations to confirm theadvantages of using Ag-NPs for root canal disinfection.
Two main issues associated with Ag-NPs are the potential discol-oration of dentin and toxicity toward mammalian cells. Gomes-Filhoet al (61) tested 2 different concentrations of Ag-NPs (90mm) in Wistarrat models. The in vivo tissue reaction of a mild to moderate chronicinflammatory response was observed and was concentration depen-dent. After 15 days, 47 ppm Ag-NPs showed a reaction that was compa-rable with 2.5% sodium hypochlorite. The toxic concentrations of silverions are approximately 1–10 mg/L, and Ag-NPs are 10–100 mg/L foreukaryotic cells (62). Research has been directed toward modifyingand developing Ag-NPs with specific antibacterial activity and lowercytotoxicity to host cells (63). Other than the size and structure ofAg-NPs, the presence of proteins in a biological medium have beenshown to greatly influence the interaction and toxicity of these nanopar-ticles toward cells.

Clinical SignificanceAg-NPs with significant antibacterial activity could be used for

root canal disinfection. However, the prolonged interaction timerequired by Ag-NPs for effective bacterial killing needs to be consid-ered, and its use ideally should be limited to medicament rather thanas an irrigant. In addition, toxicity associated with silver ions shouldnot be ignored with careful selection of nontoxic concentration foruse in vivo.

Nanoparticles Incorporated Sealersand Restorative Materials

Root canal filling material is expected to occupy and seal the pre-pared canal space to prevent bacterial recontamination. One of thedesired properties of root canal sealer is antibacterial efficacy, whichcan eliminate bacteria remaining in the root canal system and preventbacterial penetration in case of leakage. Commonly used sealers areknown to possess antibacterial activity for a maximum period of1 week, with most of them showing a significant decrease in antibacte-


rial properties immediately after its setting (64–66). The addition ofNPs in sealers or restorative materials has been tried mainly toachieve reduced bacterial penetration, increased antibacterial activitywithin dentinal tubules, increased substantivity of sealers, andincreased diffusion of antibacterial activity.

Current ApplicationsThe incorporation of CS-NP and zinc oxid nanoparticles in a zinc

oxide–based root canal sealer and resin-based root canal sealerimproved the antibacterial property and ability to diffuse the antibac-terial component (34). The addition of nanoparticles did not deteri-orate the flow characteristics of the root canal sealer. DaSilva et al(67) also used CS-NP incorporated zinc oxide–eugenol sealer forfilling treated bovine root canals in vitro. The nanoparticle modifiedsealer inhibited biofilm formation with a significantly lower percent-age of biofilm covering the sealer-dentin interface. Another recentstudy added CS-NPs to epoxy resin sealer (ThermaSeal, York, PA), re-sulting in enhanced antibacterial ability by direct-contact andmembrane-restricted tests (68). The CS-NP incorporation signifi-cantly increased the antibacterial efficacy of these different root canalsealers even after a 4-week aging time with lesser biofilm formation atthe sealer-dentin interface with and without surface treatment. BAG-NP has been recommended to promote closure of the interfacialgap between the root canal walls and core filling materials (69).The combination of polyisoprene or polycaprolactone and BAG nano-particles that could create a hydroxyapatite interface and thus ulti-mately make the use of an endodontic sealer unnecessary has beensuggested. The incorporation of BAG fillers into the polymers underinvestigation made the resulting composite materials bioactive andimproved their immediate sealing ability. It was concluded that poly-isoprene and polycaprolactone composites with BAG showed prom-ising results as single root canal filling materials (69).

Quaternary ammonium polyethylenimine nanoparticles (QPEI-NPs) have been used to improve the antibacterial efficacy of variousroot canal sealers and temporary restorative materials (70–73).Barros et al (70) incorporated QPEI-NPs into AH Plus (Dentsply Inter-national, York, PA) and Pulp Canal Sealer EWT (SybronEndo, Orange,CA), which increased the wettability and surface charge of both AH Plusand Pulp Canal Sealer EWT. However, antibacterial efficacy of both thesealers was lost after 7 days of setting with and without the QPEI-NPs. Incontrast to the previous finding, another study displayed significantlyincreased antibacterial efficacy of AH Plus and QPEI-NPs (72). Simi-larly, Beyth et al (73) and Kesler Shvero et al (74) showed that theincorporation of QPEI-NPs in 2-paste epoxy-amine resin sealer resultedin the attraction of E. faecalis to the cationic surface and caused mem-brane destabilization and total inhibition of bacterial growth. Thesenanoparticles when bound to the sealer surface presented with theadvantage of lower toxicity because of the impediment of nanoparticlepenetration into eukaryotic cells (71, 73). QPEI-NPs exerted bacteri-cidal effects mainly by adsorption and penetration through the bacterialcell wall, interaction with the proteins and fat layer in the cell mem-brane, blocking the exchange of essential ions, destabilization of thecell membrane, and cell death (75).

Clinical SignificanceMost of the nanoparticles tested for root canal disinfection

depend on contact-mediated and time-dependent antibacterial activity.Thus, the incorporation of various nanoparticles into sealers or rootfilling materials significantly improved the antibacterial efficacy byinhibition of bacterial biofilm formation on the surface as well as theresin-dentin interface. The limited in vitro studies on nanoparticle-


Figure 3. (A and B) Transmission electron microscopic images for planktonic E. faecalis after treatment with CSRB-NPs for 15 minutes. Aggregates of CSRB-NPscould be seen surrounding the bacterial cell. (C) Nanoparticles were found attached to the bacterial cell surface and forming an envelope. The cells did not showany disruption of morphology. (D) After PDT of the sensitized bacteria, various stages of membrane damage as well as release of cell constituents were evident.(E) Scanning electron microscopic images of 3-week-old multispecies biofilms presented as a uniformly thick matlike structure covering the entire dentin surface.(Inset) Three specific bacterial morphologies are evident in higher magnification. The surface showed an abundant polymeric matrix (magnified area shown by theopen arrow). (F) CSRB-NP treatment rendered the dentin surface clean of the biofilm with open dentinal tubules. (G) RB treatment showed cleaner areas of dentinalong with dense bacterial aggregates (inset: magnified area shown by the arrowhead). (A–D, Reprinted from Nanomedicine: Nanotechnology, Biology andMedicine, volume 10, Shrestha A, Hamblin MR, Kishen A. Photoactivated rose bengal functionalized chitosan nanoparticles produce antibacterial/biofilm activityand stabilize dentin-collagen, pages 491–501, Copyright 2014, with permission from Elsevier. E–G, Reprinted from Journal of Endodontics, volume 40, ShresthaA, Kishen A, Antibiofilm efficacy of photosensitizer-functionalized bioactive nanoparticles on multispecies biofilm, pages 1604–10, Copyright 2014, with permissionfrom Elsevier.)

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1422 Shrestha and Kishen JOE — Volume 42, Number 10, October 2016

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incorporated root filling materials emphasizes thorough and standard-ized studies toward possible aspects of clinical application.
Functionalized Antimicrobial NanoparticlesThe word functionalize from Merriam-Webster dictionary

means to organize (as work or management) into units performingspecialized tasks. Functionalization could alter the surface composi-tion, charge, and structure of the material wherein the original bulkmaterial properties are left intact (76, 77). In a functionalizednanoparticle, the inorganic or polymeric materials usually form thecore substrate. Functionalized nanoparticles containing variousreactive molecules and decorated with peptides or other ligandshave led to new possibilities of combating antimicrobial resistance(9, 10) (Figs. 3A–G and 4).

Current ApplicationsNanoparticle-based photosensitizers have been considered to poten-

tiate photodynamic therapy efficacy (78, 79). Functionalized nanoparticleswith photosensitizermolecules offer unique physicochemical properties of

Figure 4. Representative field emission scanning electron microscopic images showcalcium hydroxide group, (C) MCSNs group, (D) nanosilver-incorporated MCSNs prewith Ag-MCSNs contained perforated membranes (arrows), (F and I) a bacterium in(G) nanosilver-incorporated MCSNs prepared by the template method group. (RepubliPress Ltd], from Effects of adsorbed and templated nanosilver in mesoporous calciumD, Tay FR, et al, volume 9, pages 5217–30, copyright 2014; permission conveyed th


nanoparticles, such as ultrasmall sizes, large surface area/mass ratio, andincreased physical/chemical reactivity. As mentioned in the review byKishen (13), the combination of nanoparticles with photosensitizers couldbe achieved by

1. Photosensitizers supplemented with nanoparticles2. Photosensitizers encapsulated within nanoparticles3. Photosensitizers bound or loaded to nanoparticles4. Nanoparticles themselves serving as photosensitizers (13)

The combinations of nanoparticles with photosensitizer have beenfound to enhance antimicrobial PDT (78, 80) (Table 3).

Methylene blue–loaded poly(lactic-co-glycolic) acid (MB-PLGA)nanoparticles have been tested in vitro on E. faecalis biofilm and hu-man dental plaque bacteria in combination with PDT (81). The cationicMB-PLGA nanoparticles exhibited significantly higher bacterial photo-toxicity in both planktonic and biofilm phases. It was concludedthat cationic MB-PLGA nanoparticles have the potential to be usedas carriers of photosensitizer photodynamic therapy (PDT) withinroot canals. Similarly, photosensitizer-bound polystyrene beads withrose bengal (RB) as a photosensitizer were used by Bezman et al

ing colonization of E. faecalis on root canal walls. (A) Blank control group, (B)pared by the adsorption method group, (E and H) dead bacteria in direct contactdirect contact with Ag-MCSNs showing deformed cell membrane (arrows), andshed with permission of International Journal of Nanomedicine [Dove Medical-silicate nanoparticles on inhibition of bacteria colonization of dentin, Fan W, Wurough Copyright Clearance Center, Inc.)


TABLE 3. Combinations of Nanoparticles with Photosensitizer Have Been Foundto Enhance Antimicrobial PDT Efficacy because of Several Factors

Higher concentration of photosensitizer per mass withresultant production of ROS

Reduced efflux of photosensitizer from the target cell, therebydecreasing the possibility of drug resistance

Possibility of targeting the bacteria because of greaterinteraction associated with the surface charge

Greater stability of photosensitizers after conjugationLess physical quenching effect because of photosensitizer

aggregationControlled release of ROS after photoactivation possible

PDT, photodynamic therapy; ROS, reactive oxygen species.

Data from Perni S, Prokopovich P, Pratten J, et al. Nanoparticles: their potential use in antibacterial

photodynamic therapy. Photochem Photobiol Sci 2011;10:712–20; and Guo Y, Rogelj S, Zhang P.

Rose Bengal-decorated silica nanoparticles as photosensitizers for inactivation of gram-positive

bacteria. Nanotechnology 2010;21:065102.

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(82) after activation with light, improving bacterial elimination withreactive oxygen species. However, the binding of RBwith silica nanopar-ticles resulted in a slower yield of reactive oxygen species comparedwith the free RB (80). The slower decay has been suggested to be ad-vantageous in certain applications when light/dose fraction and/ordeeper tissue or bacterial penetration is required (80).

The application of naturally occurring biopolymers such as chito-san could circumvent such issues of biocompatibility (83, 84) and favorintimate contact between the photosensitizer and the aqueoussuspension of microorganisms. In RB photosensitizer functionalizedCS-NPs, singlet oxygen released by the photoactivation of the photosen-sitizer potentiated the antibacterial activity of CS-NPs (85, 86). Shresthaet al (85) used RB to functionalize CS-NPs (CSRB-NPs), whichpossessed the combined properties of chitosan and RB. These function-alized CSRB-NPs interacted greatly with bacteria and biofilms, leading tomembrane damage and leakage of cellular constituents (Fig. 3). Theincreased uptake into biofilms of CSRB-NPs compared with the photo-sensitizer alone resulted in significant elimination of biofilm bacteriaand disruption of the structure after photoactivation. Multispecies bio-films of Streptococcus oralis, Prevotella intermedia, and Actino-myces naeslundii grown on dentin sections were completelydisrupted with a reduction in viable bacteria and biofilm thickness afterCSRB-NP and PDT treatment (Fig. 3) (86). The CSRB-NPs were found tomaintain the antibacterial efficacy in the presence of bovine serumalbumin, which is a strong tissue inhibitor (87). The higher affinityof cationic CS-NPs to bacterial cell surfaces and singlet oxygen releaseafter photoactivation of RB provided a synergistic antibacterial mecha-nism for CSRB-NPs. Furthermore, CSRB-NPs could be used for the treat-ment of infected dentin because it showed a collagen cross-linkingability that could prevent degradation of the dentin matrix. Lipopolysac-charide, the toxic component of gram-negative bacteria, was inactivatedby photoactivated CSRB-NPs as shown by reduced inflammatorymarkers in macrophage cells (88). CSRB-NPs provided a single-steptreatment of infected root dentin by combining the properties of chito-san and that of the photosensitizer to eliminate bacterial biofilms.

Fan et al (42, 89) used Ag-MBG powders to eliminate E. faecalisbiofilms of 4 weeks inside root canals. Ag-MBG showed Ag-NPs incor-porated in the mesopores that resulted in sustained release of Ag ions.Compared with unmodified mesoporous bioactive glass, Ag-MBGsshowed a significant structural disruption of biofilms. The same groupof investigators have also used mesoporous calcium silicate nanopar-ticles (MCSNs) with nanosilver incorporation (Ag-MCSNs) (90).These modifications were prepared using both the adsorption andtemplate methods. Ag-MCSNs exhibited a sustained release of Agions over time. Planktonic E. faecalis was killed by Ag-MCSNs and


showed significantly better antibacterial effects when comparedwith unmodified MCSNs. When dentin surfaces were treated withAg-MCSNs, they aggregated on the dentin surface of the root canalwalls and infiltrated into dentinal tubules after ultrasound activation(89, 90). This dentin surface treatment resulted in the significantinhibition of adherence and colonization of E. faecalis on dentin.The results of the present study indicated that nanosilver couldbe incorporated into MCSNs using the template method. Thetemplated nanosilver released silver ions, inhibited the growth andcolonization of E. faecalis both in the planktonic form and asbiofilms on dentin surfaces, and showed lower cytotoxicitycompared with Ag-MCSNs (adsorbed nanosilver). PLGA polymer so-lution containing zinc oxide, Ag, and zinc oxide/Ag nanoparticles havebeen tested to decontaminate root canals with E. faecalis (91). Theminimum colony-forming unit counts were observed in the sodiumhypochlorite groups followed by zinc oxide/Ag and Ag groups. Despitethe not so strong bacterial model, the nanoparticles containing poly-mer solutions could not achieve total bacterial elimination.

Clinical SignificanceFunctionalization of various nanoparticles showed an overall in-

crease in efficacy along with rapid action against bacteria. The photo-sensitizer functionalized nanoparticles could be used as a final rootcanal disinfection strategy, whereas functionalized BAG and AG-NPs stillneed to be considered only as long-term root canal medicament.Optimization of the concentration and applicationmethod of these func-tionalized nanoparticles is under constant progress.

ConclusionThe therapeutic efficacy of antibacterial nanoparticles necessitate

optimization of their physical, chemical, and biological characteristics,keeping in mind the tissue-specific factors at the site of infection and themethod to deliver the nanoparticles effectively in the target tissue.Nanoparticle-based treatment strategies have the potential to improveantibacterial/antibiofilm efficacy in endodontics. Functionalized nano-particles via surface modifications would provide the opportunity todeliver drugs/chemicals to the site of infection in order to selectivelyinteract with biofilm and bacteria. Newer multifunctional nanoparticlesare being developed based on the clinical requirements in collaborationwith engineers, clinicians, and biologists. In terms of toxicity of nano-particles, safe usage guidelines should be developed that could be ex-pended toward growing areas of newer antibacterial nanoparticles. Thiswhole concept of nanoparticles in health care and endodontics shouldbe accepted with positive zeal and caution in future developments.

AcknowledgmentsThe authors deny any conflicts of interest related to this study.

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