Targeted Drug-Carrying Bacteriophages as Antibacterial Nanomedicines�
Iftach Yacoby, Hagit Bar, and Itai Benhar*Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences,
Tel-Aviv University, Ramat Aviv 69978, Israel
Received 5 February 2007/Returned for modification 15 March 2007/Accepted 19 March 2007
While the resistance of bacteria to traditional antibiotics is a major public health concern, the use of extremelypotent antibacterial agents is limited by their lack of selectivity. As in cancer therapy, antibacterial targeted therapycould provide an opportunity to reintroduce toxic substances to the antibacterial arsenal. A desirable targetedantibacterial agent should combine binding specificity, a large drug payload per binding event, and a programmeddrug release mechanism. Recently, we presented a novel application of filamentous bacteriophages as targeted drugcarriers that could partially inhibit the growth of Staphylococcus aureus bacteria. This partial success was due tolimitations of drug-loading capacity that resulted from the hydrophobicity of the drug. Here we present a novel drugconjugation chemistry which is based on connecting hydrophobic drugs to the phage via aminoglycoside antibioticsthat serve as solubility-enhancing branched linkers. This new formulation allowed a significantly larger drug-carrying capacity of the phages, resulting in a drastic improvement in their performance as targeted drug-carryingnanoparticles. As an example for a potential systemic use for potent agents that are limited for topical use, wepresent antibody-targeted phage nanoparticles that carry a large payload of the hemolytic antibiotic chloramphen-icol connected through the aminoglycoside neomycin. We demonstrate complete growth inhibition toward thepathogens Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli with an improvement in potency by afactor of �20,000 compared to the free drug.
The increasing development of bacterial resistance to tradi-tional antibiotics has reached alarming levels (2, 3), spurring astrong need to develop new antimicrobial agents. Classicalshort-term approaches include chemical modification of exist-ing agents to improve potency or spectrum. Long-term ap-proaches rely on bacterial and phage genomics to discover newantibiotics that attack new protein targets which are essentialto bacterial survival and therefore with no known resistance (1,8). In both traditional and newly developed antibiotics, thetarget selectivity lies in the drug itself, in its ability to affect amechanism that is unique to the target microorganism andabsent in its host. As a result, a vast number of potent drugshave been excluded from use as therapeutics due to low selec-tivity. This brings to mind the limited selectivity of anticancerdrugs and recent efforts to overcome it by developing targetedtherapeutic strategies. Antibody-based targeted drug deliveryapproaches have been developed since the advent of monoclo-nal antibodies (6). Since then, monoclonal antibodies and de-rived single-chain antibodies were used to deliver potent cyto-toxic components to cancer cells that, once bound, internalizeand kill the target cell (7, 12). A similar immunotargeting ofbacteria is not feasible due to the lack of a bacterial internal-ization process, making the use of an extracellular releasemechanism necessary for a targeted antibacterial approach.Moreover, in comparison to cancer internalized targeting de-vices such as immunotoxins and immunoconjugates, commonantibiotics are less-potent drugs in which a threshold numberof several thousands of molecules are needed to inhibit or kill
a single bacterium. Thus, a targeted antibacterial platformshould have a significantly larger drug-carrying capacity thanan anticancer one.
Filamentous bacteriophages (phages) are the workhorse ofantibody engineering and are gaining increasing importance innanobiotechnology (9). Here we present targeted, drug-carry-ing phages as a platform for targeting pathogenic bacteria. Dueto genetic and chemical modifications, these phages representa modular targeted drug-carrying platform of nanometric di-mensions where targeting moieties and conjugated drugs maybe exchanged at will.
Recently, we have shown the feasibility of using phages astargeted antibacterial drug carriers (13). In our system,chloramphenicol (which is rarely used to treat patients sys-temically due to toxicity) was attached as a prodrug to p8coat protein molecules on the surface of filamentous phage.The phages were then targeted to bind to pathogenic bac-teria and, upon release of active chloramphenicol, retardedbacterial growth. The reported system had a limited capacityfor inhibition of bacterial growth due to a limited armingcapacity of less than 3,000 drug molecules/phage. We havenow overcome this limitation by designing a unique drugconjugation chemistry, comprising the use of (hydrophilic)aminoglycoside antibiotics as branched, solubility-enhanc-ing linkers. By changing the arming chemistry and a modi-fication of the antibody-phage conjugation method, our sys-tem, as illustrated in Fig. 1, was transformed into a viableand versatile tool for the targeting of a broad range ofpathogenic bacteria.
MATERIALS AND METHODS
All the chemicals used were of analytical grade and were purchased fromSigma (Israel). Unless stated otherwise, reactions were carried out at roomtemperature (about 22°C).
* Corresponding author. Mailing address: Green Building Room202, Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel. Phone: 972 3 6407511. Fax:972 3 6409407. E-mail: [email protected].
FIG. 1. Schematic representation of drug-carrying bacteriophages. (a) Drawing of a single fUSE5 ZZ-displaying bacteriophage. Small turquoisespheres represent major coat protein p8 monomers. Purple sphere and sticks represent the 5 copies of minor coat protein p3, which is fused toa three-color helix representing the IgG binding ZZ domain. The Y-shaped structure represents complexed IgG. (b) The red helix (representedby ribbons) represents a partial structure of a major coat protein p8 monomer, conjugated through 3 N-terminal carboxyl side chains (aspartylresidues, represented by (balls and sticks) to 3 molecules of neomycin (black). Each neomycin molecule is conjugated through a labile ester bondlinker (blue) to a molecule of chloramphenicol (red). This represents conjugation at minimal capacity (not using other free amine groups ofneomycin).
Synthesis and evaluation of chloramphenicol prodrug. A chloramphenicolprodrug, where chloramphenicol is linked through a labile ester bond to anN-hydroxysuccinimide (NHS)-ester for conjugation to amine groups was pre-pared and evaluated as described previously (13). The drug release rate whenserum esterases are used is about 15% of the conjugated chloramphenicolreleased from the carrier after 1 h of incubation in serum at 37°C with linearkinetics (13).
Preparation of ZZ domain-displaying phages. Phage fUSE5-ZZ, which dis-plays the Fc-binding ZZ domain of protein A on all copies of the phage p3 minorcoat protein, was constructed as described previously (13).
Preparation of phages for drug conjugation. fUSE5-ZZ filamentous phages(13) were routinely propagated in DH5� F� cells using standard phage tech-niques as described previously (5). Phages were usually recovered from overnight1-liter cultures of carrying bacteria. The bacteria were removed by centrifuga-tion, and the phage-containing supernatant was filtered through a 0.22-�m filter.The phages were precipitated by the addition of 20% (wt/vol) polyethylene glycol8000–2.5 M NaCl, followed by centrifugation as described previously (5). Thephage pellet was suspended in sterile Milli-Q double-distilled water at a concen-tration of 1013 PFU/ml and stored at 4°C.
Conjugation of aminoglycosides to chloramphenicol or to FITC. Solid neo-mycin or hygromycin and a stock solution of 100 �M chloramphenicol prodrugin dimethyl sulfoxide or of fluorescein isothiocyanate (FITC) in dimethyl sulfox-ide were used in all conjugations. They were mixed within 0.1 M NaHCO3, pH8.5, at a molar ratio of 1:2 for the chloramphenicol prodrug-neomycin or at amolar ratio of 1:10 for FITC-hygromycin. The reaction was stirred overnight.Next, the prepared neomycin-chloramphenicol adduct was purified by reverse-phase high-performance liquid chromatography (HPLC). A reverse-phase C18
column was used on a Waters machine with a gradient 0% to 100% of acetoni-trile (stock solution of 80% [wt/wt] in water) and water (100% water to 0%) inthe mobile phase, at a 1-ml/min flow rate. Under these conditions, the neomycin-chloramphenicol adduct eluted 18 min after sample injection, while the intactchloramphenicol-prodrug eluted 24 min after sample injection (Fig. 2a and b).Following validation by matrix-assisted laser desorption ionization–time of flightmass spectrometry (MS) (Fig. 2c), the purified neomycin-chloramphenicol ad-duct was lyophilized and conjugated to antibody-complexed phage nanoparticlesby the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) procedure.
EDC chemistry. The phage major coat protein p8 contains 3 carboxylic aminoacids (glu2, asp4, asp5) that can be conjugated by application of EDC chemistry,a rapid reaction performed at a mildly acidic pH (4.5 to 5.5) (11). Here, allconjugations were done within a total volume of 1 ml of 0.1 M Na-citrate buffer,pH 5, 0.75 M NaCl, 2.5 � 10�6 mol of the aminoglycoside, 1012 phages conju-gated to an immunoglobulin G (IgG). The reaction was initiated by the additionof 2.5 � 10�6 mol of EDC, which was repeated two more times at time intervalsof 30 min. Reactions were carried out at room temperature with gentle stirring(10 rpm) in 2-ml Eppendorf tubes for a total of 2 h. The targeted drug-carryingphage nanoparticles were separated from the reactants by two dialysis steps of16 h each against 1,000 volumes of sterile phosphate-buffered saline (PBS).
Quantifying linked chloramphenicol molecules-phage. Linked chlorampheni-col molecules-phage were quantified essentially as described previously (13).Briefly, conjugated chloramphenicol was released from the phages by incubationin rabbit serum (as a source of esterases) at 37°C for 48 h. The free chloram-phenicol was separated from the phages by using 10-kDa-cutoff ultrafiltrationcartridges (Millipore), and the number of released chloramphenicol moleculeswas calculated by using a calibration curve of free chloramphenicol absorbancethat was recorded at 280 nm.
Evaluation of serum titers by ELISA using whole bacteria as antigens. En-zyme-linked immunosorbent assay (ELISA) plates (flat bottom; Nunc, Sweden)were coated with bacteria as follows. Cells from a fresh overnight culture werecollected by centrifugation and suspended in PBS at about 108 cells/ml. Analiquot of 100 �l of the cell suspension was applied to each well of the plate thatwas spun in a centrifuge at 4,000 rpm for 5 min at 4°C. The supernatant was
carefully removed, and 100 �l of 3% glutaraldehyde in PBS was added to eachwell and left to fix the cells for 1 h. Next, the plate was blocked with 50% bovineserum. Tested sera were added in serial dilutions, the plates were incubated for1 h at room temperature (about 22°C), washed three times with PBS and incu-bated with horseradish peroxidase-conjugated goat anti-human or anti-rabbitantibodies (Jackson Immunoresearch Laboratories) for an additional 1 h. Plateswere then washed four times and developed with 3,3�,5,5�-tetramethyl benzidine(Dako). Reactions were terminated with 1 M H2SO4. The plate was read in anELISA reader at 450 nm.
Growth inhibition experiments. A 100-�l aliquot of overnight bacterial culturewas collected by centrifugation and washed in 1 ml of cold PBS. The cells werecollected again and resuspended in 100 �l cold PBS. Ten microliters of washedbacteria (107 cells) was incubated with 100 to 500 �l of targeted neomycin-chloramphenicol carrying phage nanoparticles (1010 to 1011 particles) for 1 h onice. Next, an equal volume of rabbit serum was added (100 to 500 �l) andincubated for 3 h at 37°C. One hundred to five hundred microliters of thismixture was diluted in 3 ml growth medium (Staphylococcus aureus, tryptic soybroth; Streptococcus pyogenes, Todd-Hewitt broth; Escherichia coli, 2� YT [16g/liter Bacto-tryptone, 10 g/liter Bacto-yeast extract, 5 g/liter NaCl, double-distilled water complete to 1 liter]) containing 50% rabbit serum in 13-ml tubeswith shaking at 250 rpm at 37°C. Growth was recorded by monitoring theabsorbance at 600 nm.
Calculation of potency improvement factor of targeted versus free drug. Thecalculation of the potency improvement factor is based on 1010 chloramphenicol-carrying phages, each carrying 104 drug molecules, which inhibit the growth of107 cells as effectively as 15 �g of free chloramphenicol. The percentage ofrelevant phages is based on the �5% target-specific IgG within the polyclonalserum. The estimate that 30% of the drug is released during the experiment isbased on reference 13.
RESULTS
Conjugation of chloramphenicol to phages through amino-glycoside linkers. As a solution to the limited arming effi-ciency that was mainly due to drug hydrophobicity (13), weapplied aminoglycoside antibiotics as branched solubility-en-hancing linkers. This resulted in overcoming the hydrophobicbarrier in aqueous solutions on one hand and significantlyincreased the loading potential of drug molecules per phageparticle on the other. The first example was conjugation ofchloramphenicol to the aminoglycoside neomycin. Each neo-mycin molecule has 6 primary amines, 1 of which was linked toa chloramphenicol molecule, while the other amines were leftfor further conjugation to carboxyl residues of the phage coatproteins by EDC chemistry (11) (Fig. 3a). The use of phagecoat carboxyl residues for drug conjugation instead of theamine residues multiplied the direct drug-carrying capacitypotential to �11,400 molecules per phage (3,800 p8 coatprotein copies on our fUSE5-ZZ phage with a genome sizeof 9,200 bases � 3 accessible carboxyl residues on each p8monomer).
Quantification of payload. To indirectly quantify the re-active carboxyl residues on the phage surface, we prepareda FITC-hygromycin conjugate (Fig. 3b). The aminoglycosidehygromycin is similar to neomycin in structure but differs in
FIG. 2. Reverse-phase HPLC purification and MS analysis of the chloramphenicol-neomycin adduct. (a) HPLC analysis of chloramphenicol-NHS prior to conjugation to neomycin. The chloramphenicol-NHS prodrug was separated using a gradient of acetonitrile in water on a WatersHPLC machine (RP; C18 column). Chloramphenicol-NHS eluted at 25 min postinjection. (b) HPLC analysis of chloramphenicol-neomycin adduct.The chloramphenicol-neomycin adduct was separated using a gradient of acetonitrile in water on a Waters HPLC machine (RP; C18 column). Thechloramphenicol-neomycin adduct eluted at 18 to 19 min postinjection. The collected fractions were lyophilized for further analysis by MS andconjugation to phages. (c) Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) (MS) analysis of chloramphenicol-neomycinadduct. The HPLC-purified chloramphenicol-neomycin adduct was analyzed by MS. The theoretical mass of the chloramphenicol-neomycin adduct(shown on the right of the figure) was observed by the MS analysis as a major peak at 1055.3293.
FIG. 3. Schematic representation the chemical reactions used to prepare drug for conjugation. (a) Preparation of a neomycin-chloramphenicoladduct. (1) Two chemical steps were used to modify chloramphenicol for conjugation to amine groups. In the first step, the chloramphenicolprimary hydroxyl group was reacted with glutaric anhydride to create an ester linkage, resulting in a chloramphenicol linker. In the second step,the free carboxyl group of the chloramphenicol linker was activated with NHS to allow subsequent linkage to amine groups. (2) The chloram-phenicol-NHS was reacted with neomycin in a solution of 0.1 M NaHCO3, pH 8.5, resulting in a neomycin-chloramphenicol adduct. (3) The
the number of amines groups. A hygromycin molecule con-tains 2 primary amines; one may be attached to the fluores-cent dye FITC, while the second is left for further conjuga-tion to free carboxyl residues on the phage coat by EDCchemistry. Since hygromycin serves as a direct linker, itallows a true estimation of conjugation-to-phage events. Bya linear calibration curve of fluorescence intensity as a func-tion of FITC concentration, we deduced the number ofFITC molecules and, hence, that of hygromycin moleculesthat were linked to the phages at �10,000, which is compa-rable to the number of reactive carboxyl residues per phageas calculated above.
To directly quantify the chloramphenicol that was conju-gated through the neomycin linker, we released the conjugatedchloramphenicol molecules by incubating drug-carryingphages with serum as described previously (13). We calculatedabout 10,000 chloramphenicol molecules per phage. Becauseof the additional primary amines available on neomycin, aneven larger drug payload could be obtained by manipulatingreactant concentrations and incubation times, and we couldobtain over 40,000 chloramphenicol molecules/phage withoutcompromising the phage integrity.
Improving targeting efficiency. We complexed the phageswith polyclonal antibodies that served as the targeting moietiesin our targeted drug-carrying platform. We used human seraagainst staphylococci and streptococci and a rabbit, proteinA-purified IgG against E. coli O78. All serum titers, as deter-mined by ELISA on immobilized whole bacteria, were in therange of 1:10,000 to 1:100,000 (Fig. 4). To facilitate accuratecalculation of the improvement in potency, we measured thefraction of target-specific IgGs within the sera. We found thatthe total IgG concentration in the sera was �15 mg/ml, ofwhich 4 to 5% was target specific.
In our previous study, we complexed the phages with thetargeting antibodies following drug conjugation (13). Here, we
complexed the phages with the targeting antibodies prior todrug conjugation. As a result, in addition to linking the drug tothe phage coat, the applied EDC chemistry cross-linked thetargeting antibodies to the phages (data not shown). This isimportant when considering in vivo applications, where resi-dent nonspecific antibodies may compete out the targetingantibody from the ZZ domain.
Growth inhibition of target bacteria by drug-carryingphages. We tested the ability of the targeted drug-carryingphage nanoparticles to inhibit the growth of three differentstrains of common pathogenic bacteria. Two were gram posi-tive: the methicillin-resistant Staphylococcus aureus COL and aclinical isolate of Streptococcus pyogenes. The third was a gram-negative, avian pathogenic E. coli O78 (781) (14).
The growth inhibition experiments with staphylococci weredone with the minimal amount of phages that gave totalgrowth inhibition, 1010 phage particles per 107 bacteria. Neg-ative controls were nontreated bacteria as well as bacteriatreated with nonimmune human IgG or human Fc complexedbacteriophages conjugated to the same amount of antibiotic.The results were compared to growth inhibition resulting fromvarious concentrations of free chloramphenicol. We found that1010 targeted drug-carrying phages inhibited bacterial growth,as do 15 �g of free chloramphenicol (Fig. 5a and b). Similargrowth inhibition experiments were carried out with the otherbacterial targets where we could also observe growth inhibition(Fig. 5c and d). The growth inhibition profile of SP and E. coliO78 by free chloramphenicol was similar to that of the SA cells(not shown). The partial growth inhibition observed for E. coliO78 that was treated with nontargeted drug-carrying phages(Fig. 5d) was expected due to the low level of nonspecificbinding of filamentous phages to this E. coli strain that wereported previously (13).
Calculation of potency improvement in comparison to freedrug. We calculated the potency improvement factor by as-suming that the fraction of relevant phages (that bind thetarget bacteria) is equal to the fraction of target-relevant IgGswithin the sera. As was shown in Fig. 5, 1010 targeted chlor-amphenicol-carrying phages inhibited bacterial growth as ef-fectively as did 15 �g of free chloramphenicol. Consideringthat, based on the fraction of target-specific IgG in the serum,�5% of the phages are targeted, which for 107 bacteria yields50 drug-carrying phages that actually bind each target bacte-rium. Each phage carries �104 chloramphenicol molecules, ofwhich 30% (3,000) are released during the time course of theexperiment (based on release kinetics reported in reference13). This yields 150,000 drug molecules released for each targetbacterium.
For 107 target bacteria, 15 �g of free chloramphenicol cor-responds to �3 � 109 molecules/bacterium. Hence, the po-tency improvement factor in comparison to the free drug isabout 20,000 [3 � 109free/150,000targeted].
resulting neomycin-chloramphenicol adduct was conjugated to free carboxyl groups of the phage coat by the EDC procedure in a solution of 0.1M citrate buffer, pH 5. (b) Preparation of hygromycin-FITC adduct. (1) A solution of hygromycin–0.1 M NaHCO3 was added to solid FITC,resulting in a hygromycin-FITC adduct. (2) Hygromycin-FITC was conjugated to free carboxyl groups of the phage coat by the EDC procedureas described for panel a3. The fluorescence bound to the phages was measured and calculated by a linear calibration curve of free FITC, and thenthe amount of chemical active carboxyl per phage could be calculated.
FIG. 4. Titers of the polyclonal sera. The titers of human anti-Staphylococcus aureus (SA) and -Streptococcus pyogenes (SP) sera andof rabbit anti-E. coli IgG were analyzed by ELISA with fixed bacteriaas antigens, titers above 1:50,000 were recorded for all tested sera. Serawere tested in triplicate, and error bars represent standard deviationsof the data.
The emergence of bacterial drug resistance calls for creativemeasures to overcome it. The use of some extremely potentantibacterial agents is limited by their lack of selectivity. Asolution to such a problem may be provided in the form oftargeted therapy (12). An efficient targeted drug delivery plat-form should satisfy criteria of target binding selectivity, largedrug-carrying capacity, and timely drug release at the target.
We offer targeted, drug-carrying phage nanoparticles as aversatile way to meet these criteria, as shown here with growthinhibition of both gram-positive and gram-negative pathogenicbacteria. As a model drug, we used the bacteriostatic antibioticchloramphenicol which is usually limited to topical applicationdue to toxicity to blood cells (10). The phage represents here ananometric size particle that, due to the modular assemblageof its coat, offers excellent drug-carrying capacity for its size.The arrangement of drug that is conjugated on the exterior ofthe targeted particle is unique in comparison to particulatedrug-carrying devices such as liposomes or virus-like particles.An additional innovation was introduced by the use of theamino sugar-based aminoglycosides as branched, hydrophiliclinkers, providing for the solvation of hydrophobic materialssuch as chloramphenicol, FITC, or Z-Phe (not shown). Thishad solved a major obstacle in allowing conjugation to a bio-logical entity (phage) in aqueous solutions, enabling us toconjugate a fairly large amount of hydrophobic molecules toeach phage. Indeed, we could conjugate over 40,000 chloram-phenicol molecules/phage without compromising the phageintegrity. However, working at a conjugation level of 10,000molecules/phage was sufficient to obtain complete growth in-hibition while saving on precious reagents.
The neomycin we used as the aminoglycoside linker isitself an antibiotic to which the bacteria we tested are sen-sitive. However, since it was linked to the phage coat by anonlabile bond, it could not be released and contribute tobacterial growth inhibition. One can imagine a more elegantconjugation design where the aminoglycosides are linked tothe phage by a labile bond subject to controlled release, inwhich case an additive or synergistic drug effect could havebeen obtained.
Our study demonstrated an improvement factor of 20,000 incomparison to the free drug. In our previous study, we couldshow a limited growth inhibition with a much lower potentia-tion factor. This drastic improvement can probably not beexplained on the basis of increasing drug payload from 3,000 to10,000 alone. Rather, it must be a synergistic effect resultingfrom the improved chemistry which probably affected the over-all solubility of the entire drug-carrying platform, and the newtargeting approach in which the antibodies were complexed tothe phages prior to drug conjugation with concomitant cross-linking of the antibodies to the drug-carrying phages.
Our results of growth inhibition were obtained within anartificial closed system and surely do not reflect an in vivoapplication, which will offer additional challenges to our ap-proach, as those facing other potential nanomedicines (4). Forone, the usage of polyclonal serum as we did in the presentedmodel system will not be suitable for treatment without a prioraffinity purification of bacterium-specific IgGs because we“waste” �95% of our drug-carrying phages that are not tar-
FIG. 5. Growth inhibition curves. Three strains of common patho-genic bacteria were tested: Staphylococcus aureus (SA) COL, Strepto-coccus pyogenes (SP), and E. coli O78. (a) Growth curve of SA treatedwith 1011 (filled triangles) or 1010 (filled squares) targeted drug-carry-ing phages. Controls are cells treated with or 1011 (open triangles) or1010 (open squares) drug-carrying phages conjugated to human Fc andcells treated with targeted fUSE5-ZZ phages that do not carry drug(open circles). (b) Growth of S. aureus in the presence of variousconcentrations of free chloramphenicol as follows: 20 �g, filled trian-gles; 15 �g, filled squares; 10 �g, filled circles; untreated, open circles.(c and d) Growth curves of S. pyogenes (c) and E. coli O78 (d) treatedwith 1011 (filled triangles) targeted drug-carrying phages. Controls arecells treated with or 1011 (open triangles) drug-carrying phages conju-gated to normal human IgG and cells treated with targeted fUSE5-ZZphages that do not carry drug (open circles).
geted. In fact, antibodies may not be the ideal targeting mol-ecules for drug-carrying phages because the target bacteriamay be already opsonized by patient antibodies. In such a case,one may consider other targeting approaches, as we did withpeptide-displaying phages in our previous study (13). We hopethat this work would lead to more creative and versatile meth-ods to fight our most ancient and intimate enemies, the patho-genic bacteria.
ACKNOWLEDGMENTS
We thank Ehud Gazit (Tel-Aviv University) for critical reading ofthe manuscript. We thank Marina Shamis and Doron Shabat for pre-paring the chloramphenicol-NHS compound.
I.Y. was supported by a Ph.D. scholarship from the Gertner Re-search Institute for Nanomedical Systems, Tel-Aviv University, and bythe Dan David scholarship for young scholars in future dimension.
REFERENCES
1. Barrett, J. F. 2005. Can biotech deliver new antibiotics? Curr. Opin. Micro-biol. 8:498–503.
2. Bax, R. P., and N. Mullan. 1999. Response of the pharmaceutical industry toantimicrobial resistance. Balliere Clin. Infect. Dis. 5:289–304.
3. Coates, A., Y. Hu, R. Bax, and C. Page. 2002. The future challenges facing thedevelopment of new antimicrobial drugs. Nat. Rev. Drug Discov. 1:895–910.
4. Duncan, R. 2006. Polymer conjugates as anticancer nanomedicines. Nat.Rev. Cancer 6:688–701.
5. Enshell-Seijffers, D., and J. M. Gershoni. 2002. Phage display selection andanalysis of Ab-binding epitopes, p. 9.8.1–9.8.27. In J. C. Colligan (ed.),Current protocols in immunology. John Wiley & Sons, Inc., Hoboken, NJ.
6. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secret-ing antibody of predefined specificity. Nature 256:495–497.
7. Kreitman, R. J. 1999. Immunotoxins in cancer therapy. Curr. Opin. Immu-nol. 11:570–578.
8. Payne, D., and A. Tomasz. 2004. The challenge of antibiotic resistant bac-terial pathogens: the medical need, the market and prospects for new anti-microbial agents. Curr. Opin. Microbiol. 7:435–438.
9. Sarikaya, M., C. Tamerler, A. K. Jen, K. Schulten, and F. Baneyx. 2003.Molecular biomimetics: nanotechnology through biology. Nat. Mater. 2:577–585.
10. Skolimowski, I. M., D. A. Rowley, R. C. Knight, and D. I. Edwards. 1981.Reduced chloramphenicol-induced damage to DNA. J. Antimicrob. Che-mother. 7:593–597.
11. Staros, J. V., R. W. Wright, and D. M. Swingle. 1986. Enhancement byN-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated cou-pling reactions. Anal. Biochem. 156:220–222.
12. Trail, P. A., H. D. King, and G. M. Dubowchik. 2003. Monoclonal antibodydrug immunoconjugates for targeted treatment of cancer. Cancer Immunol.Immunother. 52:328–337.
13. Yacoby, I., M. Shamis, H. Bar, D. Shabat, and I. Benhar. 2006. Targetingantibacterial agents by using drug-carrying filamentous bacteriophages. An-timicrob. Agents Chemother. 50:2087–2097.
14. Yerushalmi, Z., N. I. Smorodinsky, M. W. Naveh, and E. Z. Ron. 1990.Adherence pili of avian strains of Escherichia coli O78. Infect. Immun.58:1129–1131.
