REVIEW

10.2217/17435889.3.3.329 © 2008 Future Medicine Ltd ISSN 1743-5889 Nanomedicine (2008) 3(3), 329–341 329

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Antibacterial nanomedicineIftach Yacoby1,2 &Itai Benhar1,3† †Author for correspondence1Department of Molecular Microbiology & Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, IsraelTel.: +972 3640 7511;Fax: +972 3640 9407;2E-mail: yacobyif@post.tau.ac.il 3E-mail: benhar@post.tau.ac.il

Keywords: antibiotic resistance, antibody–drug conjugates, antiseptics, target-specific peptides, targeted therapy

Recent advances in the field of nanotechnology led several groups to recognize the promise of recruiting nanomaterials to the ongoing bat tle against pathogenic bacteria . A large battery of newly discovered and developed nanomaterials has been accumulating during the last decade , therefore , it could be anticipated that it should only be a mat ter of t ime until such preliminary nanomedicine applications are presented . We review some of these pioneering studies in which nanomaterials have been evaluated as potential therapeutics, antiseptics or disin fectants. These studies can be divided roughly into two groups. The first are studies o f antibacterial nanomedicines that are based solely on synthetic (artificial) materials. The second group comprises studies o f antibacterial nanomaterials that are based on biological substances used in their natural or in a modified form . We will discuss the physicochemical and antibacterial highlights o f each material and present the future perspectives o f this emerging field .

Nanomedicine is considered as the use of mole-cular devices to address medical problems [1]. Asa broader definition, the term nanotechnologyincludes ‘materials that at least one of theirdimensions that affects their function is in thescale range between 1 and 100 nm’ [1]. Theapplication of nanomaterials against pathogenicbacteria is a crucial step in the ongoing battleagainst these microorganisms because theincreased development of bacterial resistance totraditional antibiotics has created a great needfor the development of new antimicrobialagents. The application of nanomaterials as newantimicrobials should provide novel modes ofaction and/or different cellular targets comparedwith existing antibiotics. The unmet medicalneed for new antibiotics, coupled with revolu-tions in genomics, high-throughput screeningand medicinal chemistry, has already spurred thedrug industry to search for totally new agentsthat are effective in the treatment of bacterial dis-ease caused by resistant organisms [2] As a result,new classes of compounds designed to avoiddefined resistance mechanisms are undergoingclinical evaluation [3]. Examples of such relativelynovel therapies are bacteriophages [4], naturallyoccurring or synthetic antimicrobial peptides [5]

and antibacterial photodynamic therapy [6]. Thefield of nanomaterials science has also providednovel materials with unique properties that makethem suitable for the encapsulation of activemolecules that may be harnessed for antimicro-bial applications [7]. Here, we will focus on sev-eral examples of nanomedical approaches, which

have nothing in common but their nano-sizeddimensions. The presented examples are ofnanomaterials developed for medical applica-tions and others designed to be used as antisep-tics or disinfectants. We define two groups ofantibacterial nanomedicines: the first are com-pounds derived from artificial synthetic materi-als, whereas the second comprise materials basedon natural biological substances.

Synthetic antibacterial nanomedicinesCarbon nanotubes & fullerenesCarbon nanotubes (CNTs) (Figure 1A) and fuller-enes (Figure 1B) are common building blocks innanotechnology. Today, most nanotube-relatedpublications describe electronically orientedCNTs and components for microchip produc-tion methods. The first information concerningthe potential toxicity of CNTs described thepotential toxicity from exposure to single-walled nanotubes (SWNTs) and their potentialhazardous environmental impact. Such data arescarce, often debated [8] and focused on humancells [9–12].

An investigation of CNT toxicity, using anti-microbial activity as a surrogate marker, was pre-sented in a recent report by Kang et al. [13].These authors investigated the interaction ofwell-characterized, low metal content, narrowlydistributed, pristine SWNTs with a model bac-terium, Escherichia coli K12. This study pro-vided the first direct evidence that highlypurified SWNTs can exhibit strong antimicro-bial activity. The authors concluded that severe

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cell-membrane damage by direct contact withSWNTs is the probable mechanism responsible forthe toxicity to the model bacteria. Kang et al. fur-ther suggested that direct contact between cells andSWNT aggregates was essential for the inactiva-tion of the E. coli cells and that membrane destabi-lization may be a common mechanism, also sharedby nanotubes prepared from other building blocks[14,15]. The authors concluded that their findingmay be useful in the application of SWNTs asbuilding blocks for antimicrobial materials butdid not specify for what exact applications.

In a report by Lyon et al. [16], the antimicro-bial potential of another pure carbon-basednanomoiety, the fullerene C60, was evaluated.The discovery of fullerenes in 1985 and thedevelopment of a method for their mass produc-tion in 1990 propelled an extensive field offullerene research [17,18]. Lyon et al. [16] investi-gated the potential environmental impact ofnano-C60. To that end, the authors tested theinteractions between nano-C60 and two common

laboratory bacteria, E. coli DH5D, a Gram-neg-ative bacterium, and Bacillus subtilis, a Gram-positive bacterium.

The physicochemical properties of nano-C60differ from those of pristine C60 [19]. Nano-C60 iscrystalline and the particles, which have a negativesurface charge, vary in size from 10 to over300 nm in diameter. Nano-C60 has an ultravio-let/Vis spectrum characterized by absorptionproperties of both solid C60 and C60 dissolved intoluene. Moreover, it is not possible to extractnano-C60 directly from water into toluenebecause it behaves as a stable nanoparticle suspen-sion [20]. The toxicity of C60 was reported in sev-eral papers that addressed the toxicity of C60derivatives, with conflicting results, reflecting howthe properties of the fullerene compound dependson the specific derivatives [21,22]. Other fullereneshave shown the capability to affect oxygen-radicaldamage on cultured cells. One fullerene derivativecaused lipid peroxydation [23] whereas anotherderivative was able to cause DNA cleavage [24].

Figure 1. Several potential antibacterial nanomedicines.

(A) A single-walled carbon nanotube. (B) A C60 fullerene. (C) A targeted drug-carrying phage nanoparticle. The objects are not drawn to scale.

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In the study by Lyon et al., before the toxicitytests on bacteria, the authors found that the addi-tion of salts and other media constituents affectthe size of the nano-C60 particles in water [16].Higher salt concentration correlated with a largerparticle size. By taking account of the size issue,the toxicity tests had been carried out in low saltand low protein media. The toxicity assay resultsshowed that both Gram-positive and Gram-nega-tive bacteria were affected by nano-C60, with theminimal inhibitory concentrations (MICs) andminimal bacteriocidal concentration (MBCs) ofnano-C60 against E. coli (MIC of 0.5–1.0 mg/l,MBC 1.5–3.0 mg/l) and B. subtilis (MIC of1.5–3.0 mg/l, MBC of 2–4 mg/l) being in thesame order of magnitude. These were comparablewith the MIC/MBC for carboxyfullerenereported previously for Gram-positive strepto-cocci and staphylococci [25]. In this last-citedstudy, Gram-negative bacteria were impervious tothe carboxyfullerene. The EC50 of nano-C60, astested on Vibrio fischeri, was 1.08 mg/l. In com-parison, the EC50 for the common quaternaryammonium herbicide paraquat is 603 mg/l andthe EC50 for benzene is 2 mg/l [26].

The mechanism of toxicity was not addressedexperimentally by the authors but they proposedthree hypotheses: nano-C60 disrupts electrontransport, nano-C60 punctures bacterial mem-branes or nano-C60 produces radical-oxygen spe-cies that are toxic. The authors mentioned that,in contrast with carboxyfullerene, which punc-tures the membranes of some Gram-positivebacteria but not Gram-negative bacteria [25],nano-C60 was toxic to both Gram-positive andGram-negative bacteria. Because the objective ofthe study was to evaluate the interactionsbetween C60 fullerenes and the environment, nopotential therapeutic application was actuallysuggested by the authors. The earlier study [25],in which the toxicity of carboxyfullerene toward20 bacterial isolates was evaluated, identified themechanism of toxicity as membrane destabiliza-tion and suggested that carboxyfullerenes couldbe considered as antimicrobial agents againstGram-positive cocci.

Bioactive glassesBioactive glasses of the SiO2-Na2O-CaO-P2O5system have some antimicrobial activity whensuspended in aqueous solutions through therelease of their ionic compounds over time [27].Waltimo et al. [28] studied the antimicrobialeffect of nanometric bioactive glass 45S5 that isused as a disinfectant in dentistry, the production

of which became feasible only recently [29]. Theproduction technique was based on flame-spraysynthesis that has been extended from the pro-duction of metal oxides to more complex materi-als, such as salts, and even more complex systems,such as the five element-containing bioactiveglasses [30]. Glasses prepared by flame-spray syn-thesis consist of amorphous nanoparticles with aprimary particle size of 20–60 nm and specificsurface area of 70 m2/g. In contrast to the nano-form of bioactive glass, the currently marketedbioactive glasses for dental applications are manu-factured by the melting of corresponding oxidesfollowed by grinding of the cooled product.Depending on the manufacturer, the resultingshards are sieved to obtain particles in the rangeof 10, 50 or several hundred µm, resulting in asurface area in the range of a few m2/g [31].

The release of Na+ and Ca2+ ions from, and theincorporation of H3O+ protons into, the corrod-ing bioactive glasses of the SiO2-Na2O-CaO-P2O5 results in a high-pH environment in closedsystems [32], which is not well tolerated by micro-biota [33]. Conventional bioactive glasses showsome promise as dentin disinfectants [34]; how-ever, their antibacterial efficacy in human teeth isstill inferior to that of the commonly used cal-cium hydroxide, which is referred to as the goldstandard material [35]. The results of Waltimoet al. were compared with previous attempts thatwere made to spike bioactive glass with silver toincrease its antimicrobial efficacy using E. coli,Pseudomonas aeruginosa and Staphylococcus aureusas test microorganisms. Concentrations of Ag2Obioactive glass in the range of 0.05 to 0.20 mg/mlof culture medium inhibited the growth of thesebacteria [36]. The antimicrobial properties ofcommercially available, micron-sized bioactiveglass 45S5 have been attributed to the continuousliberation of alkaline species during application,as cited before.

The study of Waltimo et al. showed that driv-ing the particles to the nanometer scale led to agreater than a ten-fold higher specific surface area,which caused the nanometric bioactive glass torelease more alkaline species and, consequently, todisplay a stronger antimicrobial effect than thecurrently applied micron-sized material. Nano-metric bioglass supernatants had a pH of 11.7,compared with 8.3 measured in counterpartsobtained from conventional bioglass suspensions.By analyzing the antimicrobial properties, nano-particulate 45S5 substantially decreased the via-bility of the pathogenic enterococci bacteria froma persisting root canal infection over time. The

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authors concluded that further studies shouldfocus on the efficacy of the nanometric bioglassmaterial in carious teeth or counterparts withinfected root canals.

Metal oxide nanoparticlesNano-MgO is a functional material that has beenused widely in various areas. In another study ofthe question ‘does size matter?’, Huang et al. [37]

investigated the effect of varying particle size onthe bactericidal activity of nano-MgO againstB. subtilis and S. aureus. The antimicrobial prop-erties of MgO were reported previously as beingcaused by the formation of superoxide anions onits surface [38,39]. Additional work by Koper andcoworkers [40] demonstrated that nano-MgOexhibits high activity against bacteria, spores andviruses after adsorption of halogen gases becauseof its large surface area, abundance in crystaldefects and positively charged particles, whichcan result in strong interactions with negativelycharged bacteria and spores.

The authors tested the bactericidal efficacy ofthe nano-MgO against both bacteria and sporesand compared it with that of nano-TiO2. Theexperimental results showed that nano-MgOparticles with different particle sizes exhibitedexcellent bactericidal efficacy (>99%) againstS. aureus ATCC 6538, which were destroyed rel-atively easily. Moreover, the bactericidal effi-ciency against B. subtilis ATCC 9372 showed ageneral increase with decreasing nano-MgO par-ticle size, from 93% when the particle size is69 nm to 97% when the particle size is 26 nm.The other particles, assigned by the authors asMgO-1 and MgO-2, had the smallest particlesize and the lowest bactericidal efficiency againstspores (a91%). Their major observation wasthat, for particles in the size range of approxi-mately 45 to 70 nm, the bactericidal efficacy ofnano-MgO increases slowly with decreasing par-ticle size. Below approximately 45 nm, however,the bactericidal efficacy shows a much strongerdependence on particle size. This correlation issimilar to the one that was observed by Waltimoet al. [28], who had shown that the number ofMg2+ ions on the surface increases rapidly withdecreasing particle size when the particle size ofMgO is less than approximately 48 nm. The lastpart of the study evaluated an application involv-ing the addition of nano-MgO to wall paint. Inthis experiment, the authors added nano-MgOwith an average particle size of 26 nm to achieve5% on weight basis to commercial phenyl-propyl-type interior wall paint and bactericidal

experiments against S. aureus ATCC 6538 andB. subtilis ATCC 9372 were carried out. Theresults were that addition of the nano-MgO gavea significant increase in bactericidal activity,particularly against B. subtilis ATCC 9372, againstwhich the unmodified paint has a bactericidalefficacy of below 90%.

To study the mechanism of toxicity, theauthors performed Fourier transform infraredspectroscopy studies to explore the bacterialmembrane for chemical modifications that mayhave resulted from contact with the nano-MgOparticles. The markedly increased intensity ofthe C–O stretching vibration suggested thatchemical changes have occurred in the proteinsin the cell wall of the bacteria. Sawai et al.reported previously that MgO is hydrated read-ily, forming a layer of Mg(OH)2 on the surface[38,39]. This layer eventually leads to the forma-tion of super oxides [38,39,41], which lead to mem-brane malfunction and cell death. The authorsconcluded that nano-MgO may be superior toTiO2 because the latter is inactive in the absenceof direct light and is ineffective against B. subtilisspores. They suggested that MgO nanoparticlesof the larger dimensions should be effective asdisinfectants within interior wall paints.

A study by Husheng et al. [42] aimed to inves-tigate the structures and antibacterial propertiesof two kinds of sterilizing nano-SiO2 particlesthat supported zinc or silver. Recently, variousmetal-containing inorganic materials have beendeveloped as antibacterials. Silver, copper, zincand other antibacterial metals, when loaded ontoinorganic carriers and released from them slowlyby design, act as inorganic disinfectants, whichare superior in terms of safety, durability andheat resistance when compared with conven-tional organic disinfectants [43]. Silver is knownto have a wide antibacterial spectrum and is rela-tively safe, which is why the development ofinorganic bactericide and disinfectant preparedby loading silver onto various inorganic carriersis receiving extensive attention [43].

The mosaic nanomaterials were synthesizedby the adsorption methodology, using two tech-niques. The first technique involved synthesizingand adsorbing silver cations onto a nano-SiO2carrier (silver-loading nano-SiO2 specimen[SLS]), whereas the second involved co-adsorb-ing zinc and silver cations onto the same kind ofcarrier (zinc–silver-loading nano-SiO2 specimen[SLZS]). So far, other inorganic compounds hadbeen used as carriers, such as zeolite, apatite,phosphates, titanium oxides and glass. The

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authors chose SiO2 because it possesses a porousstructure that can adsorb various ions andorganic molecules easily in its pores and on itssurfaces. The adsorption properties of SiO2make it one of the most promising carriers suita-ble for the development of high-performanceantibacterial and bactericidal materials.

The particle size of the SiO2 nanoparticles wasin the range of 20 ± 5 nm and special surface area(SSA): 640 ± 50 m2/g. The Ag+ and Zn2+ con-tents of SLS and SLZS specimens were approxi-mately 0.5% of Ag+ and 4–9% of zinc. Theauthors concluded that the amount of silverloaded in SLZS was similar to that in SLS. How-ever, the addition of zinc cations had no influenceon the adsorption of silver cations on the nano-SiO2 carrier.

To study the antibacterial properties of theSiO2 nanoparticles, the authors incubated E. coliand Streptococcus faecalis bacteria with 2.5 mg ofSLS or SLZS and made live counts at varioustime points. They observed that, for E. coli, thecontact time needed to kill all the viable cells was6 h with SLS and 4 h with SLZS, whereas, forS. faecalis, the same effect was obtained after con-tact with SLS for 10 h and SLZS for 6 h. Theseresults led them to conclude that the antibacterialeffect of SLZS was superior to that of SLS. Theysuggested that it was because SLZS containedZn2+ besides Ag+ and, consequently, more anti-bacterial active sites than SLS, resulting in accel-erated absorption speed and improved adsorptioncapability of SLZS. At the same time, the chanceof the antibacterial active sites being in contactwith bacteria was enhanced, so the speed of anti-bacterial process became faster [44]. The authorsconcluded that SLZS and SLS metal-releasingSiO2 carriers should be useful when incorporatedas disinfectants in dental resin-based materials toprovide antibacterial activity.

In an additional report concerning encapsu-lated silver, Melaiye et al. [45] presented the fabri-cation of antimicrobial electrospun Tecophilic®

nanofibers. These nanoparticles encapsulate thetoxic material silver(I)-imidazole cyclophane. Inthe last two decades, cyclophane compoundsreceived much attention in the development ofsupramolecular chemistry [46]. Previous reportsby the authors group [47] and others [48] describethe synthesis and structural analysis of imidazo-lium cyclophanes and their metal complexes.Moreover, the advances of using silver in wound-care management are not only focused on theantimicrobial effect of silver on chronic ulcers,extensive burns and difficult-to-heal wounds,

but also on the convenience of application,patient comfort and sustained release of silverions with increased concentration at the woundsurfaces [49,50]. The authors tested their antimi-crobial silver-based compound, silver(I)-N-pincer 2,6-bis(hydroxylethylimidazolemethyl)-pyridine hydroxide, a water-soluble silver(I)carbene complex, on some clinically importantmicroorganisms; three bacteria: E. coli,P. aeruginosa and S. aureus; and three yeast orfungi: Candida albicans, Aspergilus niger and Sac-charomyces cerevisiae [51]. The authors encapsu-lated silver(I) N-heterocyclic carbene complexesby electrospinning [52], which is a versatilemethod for the fabrication of fibers with dia-meters ranging from a few nanometers to severalmicrometers. By creating an electrically chargedjet of polymer solution or polymer melt, thepolymer is elongated and solidifies. The resultingfibers are used or have the potential to be used infilters, coating templates, protective clothing,biomedical applications, wound dressing, drugdelivery, solar sails, solar cells, catalyst carriersand reinforcing agents for composites [53,54].

Tecophilic is a family of hydrophilic polyether-based thermoplastic aliphatic polyurethanes. It isa medical-grade polymer capable of absorbingwater content up to 150% of the weight of its dryresin. Tecophilic was used as the polymer forencapsulating the silver complex because it can beelectrospun from ethanol and has excellenthydrophilic properties. Excellent hydrophilicproperties are important because water is neces-sary to facilitate the release of silver ions from theencapsulated silver complex in the polymermatrix. It is also envisaged that Tecophilic willmaintain a moist environment around the woundbed because it is known that good hydration isessential for optimal wound healing [55].

Melaiye et al. reported the measured thicknessof the fiber mat as measured by scanning-elec-tron microscopy with pure Tecophilic (100 µm),25:75 silver complex/Tecophilic (30 µm) and75:25 3/Tecophilic (60 µm), respectively. Next,the antibacterial properties were tested and thebactericidal activity showed a clear zone of inhi-bition within and around the fiber mat after anovernight incubation of the agar plate at 35°C.The fungicidal activity was observed after 48 h ofincubation at 25°C. Pure Tecophilic fiber matthat was used as a control had no growth-inhibi-tory effect. Additional bacteriostatic activity wasobserved for S. aureus and E. coli after 10 days ofthe daily streaking of the LB broth solution on anagar plate. Visual inspection of the incubated

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solutions showed no growth of the organism. Bycomparing the bactericidal effect of encapsulatedversus free silver it was found that fiber mat 75%(silver/Tecophilic) with 424 µg/ml of silver haseightfold lower silver concentration thanAgNO3 (3176 µg/ml) and has a faster killingrate then silver nitrate. Finally, the assessment ofthe acute toxicity of the ligand on rats showed aLD50 of 100 mg/kg of rat, a value considered tobe moderately toxic.

To conclude, the encapsulation of the silverheterocyclic carbene complexes increases the bio-availability of active silver species while reducingthe amount of silver used. Encapsulating silver(I)carbene complexes in nanofibers are promisingmaterials for sustained and effective delivery ofsilver ions with maximum bactericidal activityover a longer period of time than aqueous silver.The intended application is primarily as part ofwound- or burn-dressing materials.

Bio-inspired antibacterial nanomedicinesBiopolymersThe following studies describe the applicationof nanoparticles of biological origin in theirnatural or in a modified form as antibacterials.The first example involves an additional appli-cation of electrospun nanofiber mats; however,in this case, they were spun from the polysac-charide chitosan. Polymers with intrinsic bacte-riostatic and/or bactericidal activity and, inparticular, polysaccharides are considered aspromising for wound-healing and -dressingapplications. The natural polysaccharide chi-tosan was reported to possess advantageous bio-logical properties, such as hemostatic activity,nontoxicity, biodegradability, intrinsic antibac-terial properties and the ability to affect macro-phage function, which contributes to fasterwound healing [56,57]. Ignatova et al. [58] studiedthe antibacterial properties of nanoparticles thatwere fabricated from quaternized chitosan(QCh) and poly(vinyl alcohol). QCh deriva-tives illustrate a higher activity against bacteria,broader spectrum of activity and higher killingrate as compared with those of chitosan [59,60].Nanofibers have been electrospun successfullyfrom water-soluble nonionogenic polymers, suchas polyoxyethylene [61] and poly(vinyl alcohol)(PVA) [62]. PVA is interesting for wound treat-ment because it is highly hydrophilic, has aninherent fiber- and film-forming ability and canbe cross-linked easily. The cross-linking of electro-spun PVA mat in the solid state has been reported

[9,63]. After immobilization of model drugs (8-hydroxyquinoline derivatives) in chitosan- orN-carboxyethylchitosan-containing nanofibers,they acquire antimicrobial and antimycoticactivity [64,65]. They intended to study the prepa-ration of QCh-containing nanofibers by electro-spinning of mixed QCh/PVA aqueous solutions,as well as the antibacterial properties of photo-cross-linked electrospun QCh/PVA mats.

Ignatova et al. reported that the measuredaverage diameters of the QCh/PVA fibers were600 and 145 nm for total polymer concentrationof 13 and 8 wt%, respectively. The free form ofQCh was reported to show high antibacterialactivity against S. aureus [66]. In the currentstudy, the antibacterial activity of photo-cross-linked electrospun QCh/PVA mats was evalu-ated against S. aureus and E. coli. Toxicity wastested by the viable cell-counting method.S. aureus bacteria were killed within 60 min ofcontact with cross-linked QCh/PVA electrospunmat containing 2845 µg/ml QCh, in contrast toelectrospun photo-cross-linked PVA mat, used asa control, which did not modulate bacterialgrowth. The electrospun QCh/PVA mats werealso exposed to the Gram-negative bacteriaE. coli. A reduction of bacterial growth by 98%was observed after 120 min exposure to thephoto-cross-linked QCh/PVA nanofibers.

To conclude, the electrospun QCh/ PVA fib-ers had diameters of 60–200 nm with narrowdiameter distribution. Moreover, the higher thecontent of QCh, the smaller was the diameter ofthe nanofibers. The electrospun QCh/PVAnanofibrous mats were stabilized successfullyagainst dissolution in the aqueous environmentusing photo-mediated cross-linking. Finally,photo-cross-linked electrospun nanofibrousQCh/PVA mats had good bactericidal activityagainst the Gram-negative bacteria E. coli andalso against the Gram-positive bacteria S. aureus.

In another bio-inspired study by Salmasoet al. [67], the fabrication of nisin-loaded poly-L-lactide nanoparticles and their antimicrobialactivity was reported. Nisin belongs to the lanti-nobiotics, which are a family of antimicrobialproteins of bacterial origin containing unusualamino acids, such as lanthionine [68]. These bac-teriocins display a broad inhibitory spectrumagainst a variety of Gram-positive bacteria.Recently, the US FDA recognized nisin, a bacte-riocin produced by Lactococccus lactis, as a foodadditive [69]. However, the use of nisin in foodpreservation is strongly limited by its structuralinstability, deprivation by interaction with food

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and cell matrixes and development of tolerantand resistant Listeria [70]. Therefore, usage ofexcessive amounts of nisin is required to guaran-tee effective pathogen growth inhibition. Nisin-loaded polymeric micro- and nanoparticles seemto be promising formulations to achieve long-lasting antimicrobial activity. Poly-L-lactide nan-oparticle systems have been investigated activelyas protein-drug-delivery systems because theycan enhance the biological performance of bio-active molecules [71–73]. With respect to otherslow-release systems, polymeric micro-/nano-colloids are stable physically and can be formu-lated easily with a variety of materials, obtaininga controlled rate of drug release. Polymeric-drug-delivery systems are widely evaluated in the fieldof oncology [74] and here we see the first exampleof an antibacterial application of such systems.

Salmaso et al. dealt with the preparation ofstable long-lasting antimicrobial nisin-loadedpolymeric micro-/nanoparticles, which can bedispersed in food, pharmaceutical products orother materials with different physical consist-ence. Nisin–poly-L-lactide (PLA) nanoparticleswere prepared from the peptide nisin and PLAby using the supercritical CO2 in mixed solventmethod [75]. This material was chosen owing toits biodegradable and nontoxic characteristic,which is common in protein formulation [76].Furthermore, PLA possesses suitable propertiesfor compressed CO2 antisolvent precipitationthat was used for fabrication. The observedparticle size was in the range of 250 to 400 nm.

Next, the antimicrobial properties were testedagainst Lactobacillus. A total of 1 mg of free nisinor nisin-equivalent nanoparticles (20 mg of 5%nisin A-loaded nanoparticles or 5 mg of 20%nisin A-loaded nanoparticles) was dissolved orsuspended in 5 ml of sterile MRS medium. Atotal of 1% Lactobacillus delbrueckeii spp. bul-garicus culture was added to the medium andincubated overnight, after which dilutions weremade to obtain a colony-forming unit count.The two formulations, 5 and 20% nisin-loadednanoparticles, displayed similar behavior andprolonged activity, as measure by inhibition ofbacterial growth for up to 40 days. In compari-son, free nisin samples displayed antibacterialactivity for 7 days, whereas the unloaded PLAnanoparticles had no antibacterial activity.

To conclude, the nisin-loaded nanoparticles area typical example of the great potential of thistechnique in protein formulation. Nisin-loadedpolymeric nanoparticles fabricated by the GASprecipitation technique demonstrated long-lasting

antimicrobial activity. Indeed, this formulationprovides for slow protein release and protein sta-bilization, which yields an efficient antimicrobialsystem that should prove to be useful in food andpharmaceutical preservation.

These last examples are studies by the author’sgroup that are focused at applying filamentousbacteriophages (phages) as a targeted drug-carry-ing platform [77,78]. The goal of these studies isthe fabrication of a universal, modular solutionfor the eradication of pathogenic bacteria andother cells that are bearers of disease. Phagenanoparticles can actually be described as nano-needles, with diameters of approximately 8 nm,which can be used to deliver a large payload of acytotoxic drug to the target cells (Figure 1C). Toapply this platform against pathogenic bacteria, adrug was linked to genetically modified phagesby means of chemical conjugation through anesterase-cleavable linker subject to controlledrelease by serum esterases. Thus, as long as thedrug is in a conjugated state, it is essentially aprodrug devoid of cytotoxic activity, which isactivated post cleavage from the phage in prox-imity to the target site. The guidance of thedrug-carrying phages to target cells was mediatedby genetic expression of a targeting moiety onthe phage coat. The main achievement of ourstudies was the substitution of the drug selectiv-ity itself to a target selectivity borne by the tar-geting moiety. This approach may enable the useof neglected drugs that, owing to toxicity or lowselectivity, have thus far been excluded from clin-ical use. The feasibly of this approach was dem-onstrated by using the bacteriostatic, nonpotentantibiotic chloramphenicol (which is mostlyexcluded from systemic therapeutic applicationowing to it hemolytic properties [79]) as a modeldrug. In our proof-of-concept study [78], target-ing was accomplished by using two different tar-geting moieties: target-specific peptides, selectedfrom a peptide phage-display library, which weredisplayed on the major coat protein of the phage;or antibodies linked to the phages via an IgG Fc-binding ZZ domain that is fused to the g3pminor coat protein of the phage. As a model tar-get, we used the bacterial pathogen S. aureus.The preliminary system [78] suffered from lim-ited ability to inhibit bacterial growth owing to alimited arming capacity of less than 3000 drugmolecules/phage and limited solubility of theentire platform, limitations that were mainly dueto the hydrophobicity of the drug. To overcomethese limitations, we developed a ‘second genera-tion’ of the platform by developing a unique

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337future science groupfuture science group www.futuremedicine.com

drug-conjugation chemistry, based on the appli-cation of hydrophilic aminoglycoside antibioticsas branched, solubility-enhancing linkers [77].The replacement of the arming chemistry and amodification of the antibody–phage conjuga-tion method, improved our system into a viableand versatile tool for the targeting of a broadrange of pathogenic bacteria, such as S. aureus,Streptococcus pyogenes and E. coli, each targetedby microbe-specific antibodies. Experimentally,the new drug-conjugation approach led to anarming rate of over 40,000 chloramphenicolmolecules/phage. These results had providedimpressive improvement in drug potency ofapproximately 20,000 in comparison to the freedrug, as measured by bacterial growth inhibi-tion in liquid culture. This large drug-carryingcapacity was made possible by using the exteriorof the phage coat that offers many docking sitson the 3000 coat-protein monomers. Otherphage- or virus-based drug-delivery model sys-tems pack the drug within the particle andhence have a limited drug-carrying capacity [80].

In conclusion, drug-carrying phage representsa versatile therapeutic nanoparticle that, owing tothe tailoring of its coat, by the simplicity of whichit can be equipped with a targeting moiety, andits massive drug-carrying capacity, may becomean important general targeting drug-deliveryplatform. By comparison to particulate drug-car-rying devices, such as liposomes or virus-like par-ticles, the arrangement of drug that is conjugatedin high density on the exterior of the targetedparticle is unique. A dense coating of the phagewith aminoglycosides and drugs might produceadvantages that are regarded as challenges in theapplication of phages as therapeutics; the pri-mary, of course, is the immunogenicity on in vivoadministration. Indeed, our unpublished resultshave shown that drug-carrying phages are hardlyrecognized by commercial antiphage antibodiesand generate significantly lower antiphage anti-body titers when used to vaccinate mice (in com-parison to ‘naked’ phages) [Vaks et al., Unpublished Data].Further, we found that drug-carrying phages arenontoxic to BALB/c mice up to a high dose of1012 phage particles injected intravenously orintraperitoneally. The therapeutic capacities oftargeted drug-carrying phages in mouse modelsare being studied currently.

ConclusionThis review presents nine nanomedicine-ori-ented applications of antiseptics, disinfectantsand antibacterial therapeutics, which are sum-

marized in Table 1. The first six studies describesynthetic nanomaterials with antibacterial activ-ity. The first describes CNTs that can providestrong antimicrobial activity, probably mediatedby membrane damage and subsequent cell inac-tivation [13]. The second study is an investigationof the potential environmental impact of nano-C60. The toxicity assay results showed bothGram-positive and Gram-negative bacteria [16].It suggested that nano-C60 may function bepuncturing bacterial membranes or by produc-ing radical-oxygen species that are toxic. Thethird study was of bioactive glasses of the SiO2-Na2O-CaO-P2O5 system [28]. The study by Wal-timo et al., focusing on the potential dentistryapplications, showed that driving the particles tothe nanometer scale led to a greater than ten-fold higher specific surface area, which causedthe nanometric bioactive glass to release morealkaline species and, consequently, to display astronger antimicrobial effect. The fourth studyof the antimicrobial properties of MgO, with thepotential application as a wall-paint additive [37],showed that, for particles in the size range ofapproximately 45 to 70 nm, the bactericidal effi-cacy of nano-MgO increases slowly withdecreasing particle size. The study suggests a tox-icity mechanism of MgO involving chemicalchanges that have occurred in the proteins in thecell wall of the bacteria. The fifth study com-pared two inorganic encapsulation formats forslow release of silver [42]. One was a nano-SiO2carrier (SLS), whereas the second formatinvolved preparation by co-adsorbing zinc andsilver cations onto the same kind of carrier(SLZS). The antibacterial effect of SLZS wassuperior to that of SLS, possibly owing to thefact that SLZS contained Zn2+ besides Ag+ and,consequently, more antibacterial active sites thanSLS. The sixth study described silver-encapsulat-ing electrospun Tecophilic nanofibers [45]. Thesenanoparticles encapsulate the toxic material sil-ver(I)-imidazole cyclophane. The encapsulationof the silver heterocyclic carbene complexeswithin Tecophilic nanofibers increases the bioa-vailability of active silver species while reducingthe amount of silver used.

The last three studies involved bio-inspiredantibacterial nanomedicines; applications basedon biological substances. The first of this seriesdescribed QCh electrospun nanofiber mats [58].These electrospun QCh/PVA nanofibrous matswere stabilized successfully against dissolutionin the aqueous environment using photo-mediated cross-linking. The photo-cross-linked

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338 Nanomedicine (2008) 3(3) future science groupfuture science group

electrospun nanofibrous QCh/PVA mats had agood bactericidal activity against the Gram-neg-ative bacteria E. coli and Gram-positive bacteriaS. aureus. The next study described nisin-loadedPLA nanoparticles [67]. It showed that nisin-loaded polymeric nanoparticles fabricated by theGAS precipitation technique had a long-lastingantimicrobial activity. This formulation provides

for slow protein release and protein stabilization,which yields an efficient antimicrobial systemuseful in food and pharmaceutical preservation.

The only study that was carried out with anintention to develop a therapeutic approach wasthe last study in the review, which describestargeted drug-carrying bacteriophages [77,78].Drug-carrying phages are prepared by genetic

Executive summary

Synthetic antibacterial nanomedicines

• Carbon nanotubes (CNTs)– Single-walled nanotubes (SWNTs) can provide strong antimicrobial activity. Moreover, direct cell contact w ith highly purified,

pristine SWNTs w ith a narrow diameter distribution can cause severe membrane damage and subsequent cell inactivation.• Fullerene C60

– An investigation of the potential environmental impact of nano-C60. The toxicity assay results showed that, in both Gram-positive and Gram-negative bacteria, nano-C60 may function by puncturing bacterial membranes or by producing radical-oxygen species that are toxic.

• Bioactive glasses of the SiO2-Na2O-CaO-P2O5 system– Bioactive-glass systems have some antimicrobial activity when suspended in aqueous solutions by the release of their ionic

compounds over time. The study of Waltimo et al. showed that driving the particles to the nanometer scale led to a greater than ten-fold higher specific surface area, which caused the nanometric bioactive glass to release more alkaline species and to display a stronger antimicrobial effect.

• Nano-MgO nanoparticles– The antimicrobial properties of MgO were reported previously as being caused by the formation of superoxide anions on its

surface. For particles in the size range of approximately 45 to 70 nm, the bactericidal efficacy of nano-MgO increases slow ly w ith decreasing particle size. Fourier transform infrared analysis of the bacterial membrane suggests a toxicity mechanism involving chemical changes that have occurred in the proteins in the cell wall of the bacteria.

• Encapsulated silver– Nano-SiO2 carriers (silver-loading nano-SiO2 specimen [SLS]) were compared w ith a zinc–silver-loading nano-SiO2 specimen

(SLZS). The antibacterial effect of SLZS was superior to that of SLS, possible ow ing to the fact that SLZS contained Zn2+ as well as Ag+ and consequently had more antibacterial active sites than SLS.

• Electrospun Tecophilic® nanofibers– These nanoparticles encapsulate the toxic material silver(I)-imidazole cyclophane. The encapsulation of the silver heterocyclic

carbene complexes w ithin Tecophilic nanofibers increases the bioavailability of active silver species while reducing the amount of silver used. Encapsulated silver(I)–carbene complexes encapsulated in nanofibers were demonstrated to be promising materials for sustained and effective delivery of silver ions w ith maximum bactericidal activity over a longer period of time than aqueous silver.

Bio-inspired antibacterial nanomedicines

• Quaternized chitosan electrospun nanofiber mats– Electrospun quaternized chitosan (QCh)/poly(vinyl alcohol) (PVA) fibers had diameters of 60–200 nm w ith narrow diameter

distribution. Moreover, the higher the content of QCh, the smaller the diameter of the nanofibers. The electrospun QCh/PVA nanofibrous mats were stabilized against dissolution in aqueous environment successfully using photo-mediated cross-linking. Finally, photo-cross-linked electrospun nanofibrous QCh/PVA mats had a good bactericidal activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus.

• Nisin-loaded poly-L-lactide nanoparticles– Nisin-loaded polymeric nanoparticles fabricated by the GAS precipitation technique enable long-lasting antimicrobial activity.

This formulation provides for slow protein release and protein stabilization, which yield an efficient antimicrobial system that is useful in food and pharmaceutical preservation.

• Targeted drug-carrying bacteriophages– Drug-carrying phages are prepared by genetic engineering of filamentous phages to display a targeting moiety on their coat and

chemical modification that loads the phage particles w ith a large payload of drug. The drug is linked to the phage coat through a linker that is subject to controlled release at the target cells. Such phages represent a versatile therapeutic nanoparticle technology platform that, ow ing to the tailoring of its coat, by the simplicity of which it can be equipped w ith a targeting moiety and the massive drug-carrying capacity, may become an important general targeting drug-delivery platform.

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engineering of filamentous phages to display atargeting moiety on their coat and chemicalmodification that loads the phage particles witha large payload of drug. The drug is linked to thephage coat through a linker subject to controlledrelease at the target cells. Such phages represent aversatile therapeutic nanoparticle technologyplatform that, owing to the tailoring of its coat,by the simplicity of which it can be equippedwith a targeting moiety, and the massive drug-carrying capacity, may become an importantgeneral targeting drug-delivery platform.

Future perspectiveThis review presents several nanomedicine-ori-ented applications of antibacterial nanomateri-als. They became possible when it was realizedthat, in addition to facilitating the manipulationof material towards the nanoscale, mostly in thedirection of microchips and electronically ori-ented purposes, such materials could be of greatvalue and potential for medical and sanitaryapplications. In that sense, we should regard thestudies that are presented here and the futurethey foretell differently. Some of the applicationsare straightforward and easy to implement. Anexample for the new horizon of nanomedicine isthe fields of macro sterilization, dental fillingand use on wall surfaces, furniture and perhaps

even spoons and forks. The application wouldbegin in hospitals but would eventually reachour homes and working environment. As withthe example of fullerenes [16], antibacterial activ-ity could serve as a surrogate marker for potentialgeneral toxicity. The more exciting and morecomplex applications would be in vivo whennanomaterials will be applied as actual therapeu-tics. Of note is that, of the studies that were pre-sented here, only one evaluated the potentialtoxicity to animals or humans of these new anti-microbial entities [45] and no preclinical or clini-cal studies of nanoparticles have been carried outso far. This is an obvious result of the newlydeveloping field of nanomedicine research.

Financial & competing interests disclosure Work on targeted drug-carrying bacteriophages in theauthor’s group was supported in part by a grant from theIsrael Public Committee for Allocation of Estate Funds,Ministry of Justice, Israel, and by the Israel Cancer Associa-tion. I Yacoby was supported by a Dan David PhD scholar-ship for young scholars in future dimension. The authorshave no other relevant affiliations or financial involvementwith any organization or entity with a financial interest inor financial conflict with the subject matter or materialsdiscussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production ofthis manuscript.

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