Polydiacetylene Nanovesicles as Carriers of Natural

of Fresh Produce, Agricultural Research Organization, The Volcani Center, P.O. ... The ultimate goal of this study was developing antimicrobial fo...
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Polydiacetylene Nanovesicles as Carriers of Natural Phenylpropanoids for Creating Antimicrobial Food-Contact Surfaces Navneet Dogra,†, ⊥ Ruplal Choudhary,‡ Punit Kohli,† John D. Haddock,† Sanjaysinh Makwana,‡ Batia Horev,§ Yakov Vinokur,§ Samir Droby,§ and Victor Rodov*,§ †

College of Science, 1245 Lincoln Drive, Neckers 157A, Southern Illinois University, Carbondale Illinois 62901-4403, United States College of Agricultural Sciences, Southern Illinois University, Carbondale, Illinois 62901-4619, United States § Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel ‡

S Supporting Information *

ABSTRACT: The ultimate goal of this study was developing antimicrobial food-contact materials based on natural phenolic compounds using nanotechnological approaches. Among the methyl-β-cyclodextrin-encapsulated phenolics tested, curcumin showed by far the highest activity toward Escherichia coli with a minimum inhibitory concentration of 0.4 mM. Curcumin was enclosed in liposome-type polydiacetylene/phosholipid nanovesicles supplemented with N-hydroxysuccinimide and glucose. The fluorescence spectrum of the nanovesicles suggested that curcumin was located in their bilayer region. Free-suspended nanovesicles tended to bind to the bacterial surface and demonstrated bactericidal activity toward Gram-negative (E. coli) and vegetative cells of Gram-positive (Bacillus cereus) bacteria reducing their counts from 5 log CFU mL−1 to an undetectable level within 8 h. The nanovesicles were covalently bound to silanized glass. Incubation of E. coli and B. cereus with nanovesicle-coated glass resulted in a 2.5 log reduction in their counts. After optimization this approach can be used for controlling microbial growth, cross-contamination, and biofilm formation on food-contacting surfaces. KEYWORDS: nanoparticles, liposomes, phenylpropanoids, curcumin, methyl-β-cyclodextrin, bactericidal, Escherichia coli, Bacillus cereus, antimicrobial surfaces, glass

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INTRODUCTION Cross-contamination through processing equipment and kitchen utensils is a common dissemination path of foodborne diseases.1 The problem is aggravated by bacterial attachment to food-contact surfaces and biofilm formation. The existing chemical and physical surface decontamination measures are typically applied to the processing line before and/or after exploitation but not in the course of it. Therefore, they cannot prevent the cross-contamination event but at best reduce its risk. There is a need for additional consumer-friendly means to control microbial survival on food-contact surfaces. The concept of antimicrobial surfaces capable of inhibiting microbial pathogens and spoilage organisms has attracted attention during the past decade. It is assumed that such surface can keep itself free from microbial growth without relying solely on conventional chemical or physical intervention. Such systems were shown to be promising to control dissemination of human pathogens,2 improve food packaging,3 and effectively decontaminate water or other liquids.4 One approach to antimicrobial surfaces is based on release of active ingredients such as metal ions capable of killing or inhibiting reproduction of microorganisms.5−7 However, biocide-release coatings are suspected to contribute to the development of bacterial resistance.8 Another strategy is to bind an active ingredient to the surface and kill microorganisms on contact. For example, Madkour et al.9 described fast disinfecting surfaces functionalized with facially amphiphilic copolymers that completely killed E. coli © 2015 American Chemical Society

and S. aureus in less than 5 min. Many contact-active antimicrobial surfaces use synthetic cationic materials such as N-alkylated polyethylenimines.10,11 Lee et al.12 grew an antimicrobial polymer directly on the surfaces of glass and paper using atom transfer radical polymerization to produce nonleaching antibacterial surfaces possessing a large concentration of quaternary ammonium groups without the need to chemically graft the antimicrobial material to substrate. Poverenov et al.13 applied a direct covalent linkage of quaternary ammonium salts to prepare contact active antimicrobial surfaces on the basis of poly(vinyl alcohol), cellulose, and glass, efficient toward Bacillus cereus, Alicyclobacillus acidoterrestris, Escherichia coli, and Pseudomonas aeruginosa. The long-term effectiveness of the contact active materials was demonstrated by their repeated usage without significant loss of the antimicrobial potency.12,13 However, the drawback of these materials limiting their appropriateness to food industry is that they rely on synthetic chemicals rather than on natural foodgrade substances. Active antimicrobial surfaces and particles based on natural materials are desirable for food applications. Many natural phenolic materials possess antimicrobial properties, being at the same time nontoxic for humans, environmentally friendly, and biodegradable. Among the Received: Revised: Accepted: Published: 2557

November 12, 2014 February 19, 2015 February 20, 2015 February 20, 2015 DOI: 10.1021/jf505442w J. Agric. Food Chem. 2015, 63, 2557−2565

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Journal of Agricultural and Food Chemistry

Figure 1. Phenolic compounds tested in this study: 1, p-coumaric acid, 2, caffeic acid, 3, ferulic acid, 4, sinapic acid, 5, coniferaldehyde, 6, sinapaldehyde, 7, coniferyl alcohol, 8, sinapyl alcohol, 9, resveratrol, 10, hydroxytyrosol, 11, curcumin.

surfaces. However, curcumin impregnation conferred antimicrobial activity to wool fabric.26 Polydiacetylene (PDA) vesicles are a novel carrier system for hydrophobic materials poorly soluble in water, such as anticancer drug paclitaxel based on plant component taxol.27 Incorporation of a polymerizable diacetylene into the lipid bilayer greatly improves the stability of the vesicles compared with the conventional liposomes. Furthermore, the PDA vesicles can be covalently bound to the surface of solid substrates such as glass.28 Dogra et al.29 studied the interaction of glucose-tagged PDA vesicles (either giant or nanoscalesized) with E. coli as a model of molecular recognition mechanisms. However, to the best of our knowledge, this system has not been used so far for delivery of antimicrobial activity and for construction of antimicrobial active surfaces. The ultimate goal of this research was developing novel antimicrobial food-contact materials based on phenylpropanoids or related natural compounds. In order to reach this goal, the work comprised the following steps: (a) selecting the most promising antimicrobial compound; (b) building PDA nanovesicles functionalized with the selected compound; (c) binding the vesicles to a glass surface; (d) testing the antimicrobial activity of the vesicles, both suspended and immobilized, toward Gram-positive and Gram-negative bacteria, as well as toward yeast.

promising natural compounds are well-known plant constituents, such as resveratrol from grapes,14 curcumin from turmeric,15 hydroxytyrosol from olive,16 as well as ubiquitous lignin-related phenylpropanoids.17,18 Interestingly, the latter group acts as natural surface-functionalizing materials, impregnating the plant cell wall and conferring antimicrobial activity upon it in case of pathogen attack.19 Using natural phenolic compounds as a basis for active food-contact surfaces is still in its infancy. Rhim and Ng20 proposed to use nanocomposite technology to produce environmentally friendly antimicrobial food packaging and food-contact surfaces by blending nanolignin with biodegradable polymers. This approach proved feasible in the textile industry when fabrics with antimicrobial activity were produced by adding lignin nanoparticles to the fiber.21 To the best of our knowledge, no screening of various phenolic compounds for their potential as functionalizing ingredients for antimicrobial surfaces has been performed until now. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural phenylpropanoid dimer, is one of the promising candidates for the role of a natural food-grade surface-functionalizing phenolic compound. It is an active principle of turmeric Curcuma longa L. used as a traditional spice and folk medicine in South and South-East Asia and Middle East. Curcumin exhibits various biological activities including antiproliferative, antioxidant, and wound healing effects22 and has a self-affirmed “Generally Recognized as Safe” (GRAS) status.23 Curcumin possesses antibacterial properties against a number of Gram-positive and Gram-negative bacteria.15,24 However, poor water solubility limits the application of curcumin as antimicrobial agent in its native form. Reduction of the particle size down to the nanorange markedly improved water dispersibility and antimicrobial efficacy of curcumin.25 Nanoparticles of curcumin (nanocurcumin) having a size distribution in the range of 2−40 nm prepared by a wet-milling technique were more effective than regular curcumin against S. aureus, B. subtilis, E. coli, P. aeruginosa, Penicillium notatum, and Aspergillus niger. Transmission electron microscopy revealed permeation of nanocurcumin particles inside the Gram-positive bacteria followed by cell wall disintegration and eventually cell death. In spite of the unique combination of antimicrobial potency, food origin, and health benefits, no attempts have been taken until now to use curcumin for developing antimicrobial food-contacting

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MATERIALS AND METHODS

Chemicals. The phenolic compounds and methyl-β-cyclodextrin (MBCD, MW 1331) were supplied by Sigma-Aldrich Israel (Rehovot, Israel). If not specified differently, other solvents and chemicals were from Fisher Scientific, Fair Lawn, NJ. 1-(3-(Dimethylamino)propyl)-3ethylcarbodiimide hydrochloride and 10,12-pentacosadiynoic acid were from GFS Chemicals, Columbus OH, and 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) was from Avanti Polar Lipids, Alabaster, AL. Nutrient media were purchased from BD Difco Laboratories, Detroit, MI, except for the nutrient yeast dextrose broth (NYDB) that was prepared using nutrient broth (Difco), yeast extract (Pronadisa Conda, Madrid, Spain), and D-(+)-glucose (Merck, Darmstadt, Germany) in amounts of 0.8, 0.5, and 1.0 g per 1 L water, respectively. Screening Antimicrobial Activity of MBCD-Encapsulated Phenolic Compounds. Microencapsulation of Phenolic Compounds. The following phenolic compounds were tested in this study: p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, coniferaldehyde, sinapaldehyde, coniferyl alcohol, sinapyl alcohol, hydroxytyrosol, resveratrol, and curcumin (Figure 1). Equal volumes of a 20 mM 2558

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Figure 2. Synthesis of diacetylene monomers. Monomers: 1, 10,12-pentacosadiynoic acid, 1a, NHS-tagged diacetylene monomer, 2, glucose-tagged diacetylene monomer. ethanolic solution of a phenolic compound tested and a 20 mM aqueous solution of MBCD were mixed and stirred for 2 h on a magnetic stirrer at room temperature. Ethanol was removed from the mixture by evaporation under vacuum to half-volume (Rotavapor R124, Buchi, Flawil, Switzerland). The nonencapsulated residue of the phenolic compound was removed by filtration through a sterile 0.45 μ Millex filter with Durapore membrane (Millipore, Cork, Ireland). The obtained filter-sterilized aqueous dispersion of the phenolic−MBCD complex (Ph−MBCD) was kept in a refrigerator until use. The concentration of the phenolic compound in the Ph−MBCD was determined by diluting the aqueous dispersion with ethanol in a ratio of 1:9 and measuring its absorbance with an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences, Piscataway NJ) at a characteristic wavelength in comparison with the calibration curve of the compound in 90% ethanol. In particular, wavelengths of 262 and 425 nm were used for curcumin quantification. Assay of the Antimicrobial Activity of MBCD-Encapsulated Phenolic Compounds. Cells of E. coli (ATCC 25922) were grown overnight in LB broth (Lennox) liquid medium, separated from the medium by centrifugation, washed with sterile water, and resuspended in a fresh double-strength LB medium to reach an optical density of 0.2 at a wavelength of 600 nm. The bacterial suspensions were further diluted 1:1 with aqueous Ph−MBCD dispersions and sterile water to obtain various concentrations of phenolics but the same initial E. coli dilution. The bacterial suspensions were aseptically pipetted into the wells of a 96-well microplate, 0.2 mL per plate, and incubated for 20 h at 27 °C in an EnSpire multilabel plate reader (PerkinElmer, Waltham MA, USA) with hourly 10 s shaking. The experiments were run in triplicate and in addition to treatments (viable E. coli + Ph−MBCD) included blanks (nutrient medium without E. coli + Ph−MBCD), positive controls (viable E. coli + nutrient medium and MBCD without phenolics), and negative controls (E. coli killed by boiling + Ph− MBCD). The bacterial growth was registered by hourly measurement of turbidity as optical density at 600 nm and evaluated as added area under the growth curve (AAUC) during the whole cultivation period

(20 h). Growth inhibition was calculated as AAUC reduction (%) in the presence of a given phenolic concentration compared with a positive control without phenolics. The half-maximal inhibitory concentration (IC50) was determined as a concentration required for 50% growth inhibition and minimum inhibitory concentration (MIC) as the lowest concentration resulting in complete growth prevention. A similar technique was used for evaluating the effect of curcumin− MBCD complex on yeast Saccharomyces cerevisiae growth, except for using the NYDB instead of the LB medium. Synthesis of Diacetylene Monomers. The synthesis procedures are schematically presented in Figure 2. Synthesis of N-Hydroxysuccinimide (NHS)-Tagged Diacetylene Monomer. NHS (0.348 g, 3.0 mmol) was added to a solution of 10,12-pentacosadiynoic acid 1 (1.00 g, 2.7 mmol) in 20 mL of anhydrous dichloromethane (DCM), followed by adding 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (0.570 g, 3.1 mmol) to the same solution. The solution was stirred at room temperature for 2 h followed by rotary evaporation of the solvent. The residue was extracted with diethyl ether and washed with water three times. The organic layer was dried with magnesium sulfate for 0.5 h and then filtered. The solvent was removed by rotary evaporation to give a white solid powder of NHS-tagged diacetylene monomer 1a (1.18g, 93%). Synthesis of Glucose-Tagged Diacetylene Monomer. Synthesis of 2a. N-Boc-L-threonine (2.65 g, 12.0 mmol), acetobromoglucose (2.46 g, 6.0 mmol), and potassium carbonate (1.24 g, 9.0 mmol) were dissolved in 40 mL of dry acetonitrile under argon atmosphere with stirring for 10 min. Iodine (2.28 g, 9.0 mmol) was then added against a flow of argon. The glassware was then sealed and stirred at room temperature with the exclusion of light for 6 h. A saturated sodium thiosulfate aqueous solution was added to the above stirring solution, until the deep red color had disappeared, leaving a slightly yellow solution. The insoluble residual potassium carbonate was removed by filtration. The filtrate was concentrated to 1/4 of its original volume under reduced pressure on a rotary evaporator. DCM (40 mL) was 2559

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under vacuum. Silanization was performed at room temperature by immersing the glass slides in 4% aminosilane solution, 1% acetic acid, and 95% ethanol under continuous stirring for 4 h at room temperature. The silanized glass slides were then cleaned with ethanol for about 10 min, baked at 110 °C overnight, and brought back to room temperature. Slides were dipped in curcumin nanovesicle dispersion for 1 h and then kept in vacuum desiccator overnight. Spectroscopic Measurements and Atomic Force Microscopy. UV−vis absorption spectra of all of the samples were recorded at room temperature using a PerkinElmer Lambda 25 (spectral slit width 1 nm) UV/vis spectrometer using a cuvette of 1 cm path length. Fluorescence emission spectra of all of the samples were recorded at room temperature using a PerkinElmer Lambda 55 (spectral slit width 1 nm) luminescence spectrometer (PerkinElmer, Waltham MA, USA). The samples were excited at 350 nm, and emission was recorded in 360− 650 nm range. The topology of nanovesicles on glass surfaces was investigated by atomic force microscopy (AFM) using a TopoMetrix TMX 1010 Explorer instrument (TopoMetrix/Veeco Instruments, Santa Clara CA) in tapping mode using antimony-doped silicon tip. The largest scanning area was 80 × 80 μm, and the highest z resolution of the instrument was 0.2 nm. The samples were prepared by applying nanovesicle dispersion on a mica or glass surface, followed by overnight drying under vacuum. The transmission electron microscopy (TEM) images of liposomes were obtained after staining the liposomes with 2% phosphotungstic acid using a Hitachi S-7100 TEM instrument (Hitachi HTA, Inc., Schaumburg, IL, USA) operating at an accelerating voltage of 100 kV. The liposome samples were deposited onto Formvar-coated copper grids (Ted Pella, Inc., Redding CA, USA) for obtaining TEM images. Visualization of Nanovesicle Contact with E. coli Cells. In order to visualize the contact of nanovesicles with E. coli cells, the vesicles were tagged with Sulforhodamine 101 (SR-101) having red emission and E. coli was stained with 4′,6-diamidino-2-phenylindole (DAPI) having blue emission as described in the previous publication.29 A DAPI filter (excitation and emission band widths 349 ± 25 and 459 ± 25 nm, respectively) and a 41004 Texas Red filter (exciting and emitting band widths 527−567 and 605−682 nm, respectively) were used for capturing blue and red emission. Testing the Antimicrobial Activity of Curcumin-Functionalized Nanovesicles. The cultures of E. coli W 1485 and Bacillus cereus ATCC 14579 were grown on tryptic soy broth (TSB) medium at 37 °C for 12 h and harvested by centrifugation (Beckman J2-M1, Schaumburg, IL, USA) for 10 min at 5500g and 4 °C. The pellet was suspended in 50 mL of phosphate buffer saline (PBS) at pH 7.0. Serial dilutions were made and plated onto tryptic soy agar (TSA) plates for colony-forming units (CFU) enumeration. The antibacterial activity of suspended and glass-bound curcuminfunctionalized nanovesicles (CFN) was studied by the shake flask assay method in comparison with vesicles containing no curcumin (control). Two milliliters of the vesicle dispersion was added into the sterile conical flask containing 23 mL of PBS. For studying the antimicrobial activity of CFN-coated glass slides they were placed in a sterile conical flask containing 50 mL of PBS at pH 7.0. Glass slides coated with nanovesicles without curcumin served as control. A 1 mL amount of test culture suspension was pipetted into the flask, which brought the initial cell density of the cell suspension to approximately 105 CFU mL−1. Flasks were incubated in a gyratory water bath shaker (New Brunswick Scientific, Edison, NJ) at 36 °C for 48 h. Viable bacteria in the flasks at different time intervals were counted by plating aliquots of serial dilutions onto TSA media and incubating for 18−24 h at 35 °C. A similar approach was used for evaluating the effect of curcumincontaining nanovesicles on the survival of yeast S. cerevisiae in nongrowing medium (PBS at pH 7.4), except for using NYDB for initial growing of the S. cerevisiae culture and potato dextrose agar amended with 100 ppm chloramphenicol for plating. The incubation duration was extended to 120 h. Statistics. Bacterial counts (CFU mL−1) were obtained from three replicates, and resulting means were converted to log10 CFU mL−1.

added to the mixture, and the solution was extracted with sodium bicarbonate aqueous solution (5% w/v, 50 mL) one time, followed by brine (50 mL) two times; the organic layer was dried over magnesium sulfate. The crude products were purified by flash chromatography using a glass column packed with silica gel and a gradient solvent system hexane/ethyl acetate 10:1 to 1:4. The major fraction was collected and evaporated in vacuum to a white solid 2a; yield 1.75 g, 53%. Synthesis of 2b. The product 2a (1.5 g, 2.73 mmol) was dissolved in 20 mL of anhydrous DCM under argon. Trifluoroacetic acid (1.0 mL, 13.01 mmol) was then added dropwise to it, and the solution was stirred at room temperature for 3 h. The process was monitored by TLC until no starting materials were detected. The solvent was evaporated under reduced pressure to give a viscous liquid 2b. Synthesis of 2c. Triethylamine (1.49 mL, 10.6 mmol) and a solution of 2b (0.867 g, 1.93 mmol) in 10 mL of anhydrous DCM were added to a solution of 1a (1.0 g, 2.12 mmol) in 20 mL of anhydrous DCM. After stirring over 36 h, the solvent was removed by rotary evaporation. The residue was redissolved in 20 mL of DCM, and then it was washed with the following solutions: 1 M HCl aqueous solution (twice), saturated sodium bicarbonate solution (twice), and saturated sodium chloride solution (once). The organic layer was dried with magnesium sulfate, filtered, and evaporated to give crude semisolid. The crude products were purified by flash chromatography (silica gel, chloroform/methanol 20:1). Compound 2c was obtained as a white powder (1.102 g, 75%). Synthesis of 2. Compound 2c (1.0 g, 1.31 mmol) was dissolved in 50 mL of methanol containing 0.05 N sodium methanolate and was stirred at room temperature overnight. Ion exchange resin was added to the solution until the pH ≤ 6 was reached. The solution was filtered, and the solvent was removed by rotary evaporation to get a white solid. The crude products were purified by flash chromatography (silica gel, chloroform/methanol 2:1). Compound 2 (glucose-tagged diacetylene monomer) was obtained as a white powder (0.389 g, 50%). Preparation and Characteristic of Curcumin-Functionalized PDA Nanovesicles. Curcumin was used for preparing functionalized nanovesicles due to its superior antimicrobial potency shown in preliminary screening tests. Self-Assembly of Curcumin-Functionalized Nanovesicles. A mixture containing 10,12-pentacosadiynoic acid, glucose-tagged and NHS-tagged diacetylene monomers, and DMPC in a ratio of 4:4:2:1 was dissolved in chloroform in a round-bottom flask, and the solvent was then evaporated completely to yield a thin film of monomers. The film was hydrated with a saturated aqueous solution of curcumin and sonicated at 76 °C for 15 min using a probe sonicator (Vibra-cell Model CV 33, Sonics & Materials, Inc., Newton, CN, USA). Sonication resulted in vesicle formation and inclusion of curcumin in the hydrophobic bilayer of the vesicles. The dispersion was then passed through a 0.8 μm nylon filter (Whatman, Inc., Piscataway, NJ, USA) to remove the lipid aggregates and cooled at 4 °C overnight to promote self-assembly of the monomers. The obtained filtrate was optically clear. Free curcumin was removed by dialysis using a Spectra/ Por Biotech Cellulose Ester membrane, MWCO 100 000 (Fisher Scientific International, Inc., Hampton, NH, USA) preliminarily soaked in deionized water for 15 min. The final dispersion was collected in a vial covered with aluminum foil. Curcumin concentration in the vesicle dispersion was determined spectrophotometrically after its dilution in ratio (1:9) with ethanol using the extinction coefficient of 59 500 M−1 cm−1 in 90% ethanol at 425 nm. Typically, the curcumin concentration in the dispersion varied within the range 0.1−0.5 mM. Binding Curcumin-Functionalized Nanovesicles to Glass Surface. The nanovesicles, with or without curcumin, were bound to the surface of silanized glass slides through amidization between the amine groups of the aminosilane and the NHS head groups of PDA-NHS. Glass slides, 25 × 25 mm, were cleaned by sonication in acetone for 10 min, dried under argon for 5 min, and treated with freshly prepared Piranha solution (70% H2SO4, 30% H2O2) for 30 min. The cleaned slides were rinsed with copious amounts of distilled water and dried 2560

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Journal of Agricultural and Food Chemistry Resulting log10 CFU mL−1 reduction values were compared by analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) tests using SAS 9.2 software (SAS Institute Inc., Cary, NC).

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RESULTS Active Principle Selection: Curcumin as a Promising Antimicrobial Compound. The phenolic compounds tested varied greatly in their inhibitory activity toward E. coli. Just a few compounds having 4-hydroxy-3-methoxy substituents demonstrated a complete inhibition of E. coli growth at concentrations tested: curcumin, coniferaldehyde, and ferulic acid (Table 1). Within the series with the same substituents the Table 1. Inhibitory Activity of MBCD-Encapsulated Phenolic Compounds toward E. coli compound p-coumaric acid caffeic acid ferulic acid sinapic acid coniferaldehyde sinapaldehyde coniferyl alcohol sinapyl alcohol hydroxytyrosol resveratrol curcumin

group phenylpropanoid acid phenylpropanoid acid phenylpropanoid acid phenylpropanoid acid phenylpropanoid aldehyde phenylpropanoid aldehyde phenylpropanoid alcohol phenylpropanoid alcohol phenylethanoid alcohol stilbenoid phenylpropanoid dimer

IC50 (mM)

MIC (mM)

4-hydroxy

9.2

n.d.a

3,4-dihydroxy

8.3

>13

4-hydroxy-3methoxy 4-hydroxy-3,5dimethoxy 4-hydroxy-3methoxy 4-hydroxy-3,5dimethoxy 4-hydroxy-3methoxy 4-hydroxy-3,5dimethoxy 3,4-dihydroxy

4.4

6.4

7.9

n.d.

3.0

6.8

5.6

n.d.

13.6

n.d.

16.0

n.d.

10.5

n.d.

6.1

n.d.

0.17

0.4

substitution

3,5,4′trihydroxy 4-hydroxy-3methoxy

Figure 3. Effects of curcumin and MBCD concentrations on E. coli growth. Curves designation: (1) no additives (control); (2) 5 mM MBCD; (3) 2.5 mM MBCD; (4) 0.2 mM curcumin + 2.5 mM MBCD; (5) 0.4 mM curcumin + 5 mM MBCD; (6) 0.4 mM curcumin + 5 mM MBCD (thermally inactivated culture); (7) no additives (thermally inactivated culture). Curves 1−5 correspond to viable E. coli culture.

a

MIC was evaluated only when complete inhibition was reached; n.d., not determined.

activity declined in the order aldehyde > acid ≫ alcohol. Curcumin showed by far the highest inhibitory effect among the compounds tested. An example of the curcumin effect on E. coli proliferation is presented in Figure 3. At 0.2 mM curcumin reduced the growth rate of E. coli approximately by one-half, while 0.4 mM curcumin resulted in full arrest of bacterial proliferation, so that the growth curve overlapped with that of the dead culture. The growth of the yeast S. cerevisiae was also inhibited by the MBCD-encapsulated curcumin with an IC50 value of 0.67 mM (data not shown). No growth inhibition was shown by MBCD at 5 mM, the highest concentration tested. Curcumin was chosen as the promising active principle for further research. Characteristics of Curcumin-Functionalized Nanovesicles. Curcumin-functionalized nanovesicles (CFN) were prepared as described in Materials and Methods. The particle dimensions were in a range of 50−400 nm (Figure 4). The polymerized PDA nanovesicles containing no curcumin were characterized by absorption peaks at 590 and 640 nm, while the ethanolic curcumin solution had maximal absorption at 428 nm (Figure 5A and 5B, respectively). Both PDA and curcumin characteristic maxima were presented in the absorption spectrum of CFN (Figure 5C).

Figure 4. AFM (A) and TEM (B) images of the PDA nanovesicles.

Indication of curcumin location within the nanovesicles could be obtained from comparing the fluorescence emission spectrum of CFN with the emission spectra of curcumin in solvents of different polarities. Figure 6 represents the emission spectra of curcumin dissolved in hexane, toluene, chloroform, tetrahydrofuran, ethanol, and water whose dielectric constant values at 20 °C were 1.89, 2.38, 4.81, 7.52, 25.30, and 80.10, respectively.30 In agreement with the literature,31 polar solvents tended to shift the curcumin emission maxima toward higher wavelength. The comparison indicated that at least part of the CFN-associated curcumin was located in relatively nonpolar medium having a dielectric constant between 4.8 and 7.5, rather 2561

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than in aqueous environment. The presented dielectric constant range was comparable with that of polymerized PDA.32 It can be suggested that due to its hydrophobicity curcumin molecules tend to concentrate in the lipophilic bilayer of the nanovesicle. Interaction of Free and Surface-Bound CFN with Bacteria. When brought in contact with E. coli, the nanovesicles tended to bind to the bacterial surface as shown in Figure 7, presenting a fluorescent micrograph of SR-101-

Figure 7. Fluorescence micrograph of SR-101-tagged nanovesicles (red emission) bound to the surface of E. coli stained with DAPI (blue emission in the nucleoid region).

tagged nanovesicles (red) and DAPI-stained E. coli nucleoid (blue). As shown previously, this binding was due to the presence of covalently bound receptor (glucose) in the bilayer of the nanovesicles.29 The CFN demonstrated bactericidal activity toward both Gram-negative (E. coli) and vegetative cells of Gram-positive (B. cereus) bacteria, reducing their counts in phosphatebuffered saline (PBS) at pH 7.0 from 5 log to an undetectable level within 8 h (Figure 8). Only limited activity (not more than 1 log reduction) was observed against yeast (data not shown). The attachment of CFN to glass slides rendered antibacterial activity to their surface. Incubation of E. coli with CFN-bound glass resulted in a reduction in its viable counts by 2.5 log CFU mL−1 (Figure 9A). With B. cereus, fast viability decline was observed within 4 h of incubation with the coated glass (Figure 9B). The reduction was 1.3 log CFU mL−1 without curcumin and 2.5 log CFU mL−1 in the presence of curcumin. No significant changes in viable counts of B. cereus occurred during further incubation. In parallel, the formation of spores was observed in B. cereus cultures, both in the presence of curcumin and in the control.

Figure 5. Absorption spectra of PDA vesicles without curcumin (A), ethanolic solution of curcumin (B), and curcumin-functionalized PDA vesicles (C).

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DISCUSSION Within the series of phenylpropanoids with the same substitution pattern, the activity decreased in the order aldehyde > acid ≫ alcohol. Curcumin, with two carbonyl groups and “double coniferaldehyde” structure, also apparently obeyed this trend. Similar tendencies in the activity of phenylpropanoids against several microorganisms were demonstrated by Barber et al.,17 except for curcumin behavior that was not analyzed by those authors. Prevalence of curcumin over monomeric phenylpropanoids (e.g., ferulic acid) in anticancer and antiviral activity was demonstrated by Huang et al.33 and Bourne et al.,34 respectively. It was reported that the antibacterial effect of curcumin may be related, in particular, to inhibition of assembly and polymerization of the citoskeletal protein FtsZ playing a role in prokaryote cell division.24,35 Kaur et al.35 located the binding site of curcumin to the FtsZ molecule and stressed the potential of curcumin for the development of novel antimicrobials.

Figure 6. Emission spectra of curcumin in solvents of different polarity (top) and of the CFN-enclosed curcumin (bottom).

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Figure 8. Viable counts of E. coli (A) and B. cereus (B) in PBS. Dashed lines: free-suspended CFN (0.25 mM curcumin). Solid lines: particles without curcumin (control).

Figure 9. Viable counts of E. coli (A) and B. cereus (B) in PBS. Dashed lines: CFN-coated glass slides (approximately 1 μg curcumin per cm2). Solid lines: glass slides coated with nanoparticles without curcumin (control).

On the other hand, the shake flask assay might not ensure optimal contact of bacteria suspended in the whole PBS volume with the CFN-coated glass. These contact limitations might result in certain reduction of efficacy of glass-bound vs freesuspended CFN toward E. coli. Sporulation of B. cereus caused by contact with nanovesicle-coated glass (with or without addition of curcumin) might affect the results obtained with this species. An optimal standardized procedure for testing the antimicrobial activity of active surfaces has yet to be developed. At any rate, the trials have shown a significant count reduction of both Gram-negative and Gram-positive bacteria caused by the contact with CFN-coated glass as compared with glass coated with nanovesicles without curcumin. On the other hand, further optimization of the nanovesicle efficacy is needed. In summary, this study has demonstrated the advantages of curcumin as a potent natural antibacterial compound far exceeding other phenolic compounds tested. This activity was retained after inclusion of curcumin in liposome-type PDA nanovesicles and further covalent binding of the vesicles to glass surface. Thus, for the first time active antimicrobial surfaces functionalized by the natural food-grade compound curcumin were prepared. The activity of the curcuminfunctionalized surfaces was demonstrated toward both Gramnegative (E. coli) and Gram-positive (B. cereus) bacteria. After efficacy optimization, this approach can be implemented, without imposing a health risk, for controlling microbial growth and preventing cross-contamination and biofilm formation on food-contacting surfaces (equipment, utensils, packaging) used in the food industry and home cooking.

Our suggestion about localization of curcumin within the membrane bilayer region rather than in more polar environments of the vesicle’s cavity or interface region is in agreement with the report of Kunwar et al.36 concerning phospholipid liposomes. Similarly, Sun et al.37 investigated the interaction of curcumin with bilayer lipid membranes of giant unilamellar vesicles and came to the conclusion that curcumin initially binds to the interface and then is eventually inserted into the nonpolar hydrocarbon region. Interaction of CFN-enclosed curcumin with bacterial membranes deserves further investigation in order to understand the mode of its antimicrobial action. Several possible mechanisms were proposed for liposome−bacterial cell interaction.38 The two most relevant alternatives are (a) a fusigenic mechanism based on the merger of liposome bilayer with the cellular membrane releasing the vesicle content inside the cell38 and (b) the “collision” mechanism, including intermembrane transfer of lipophilic materials without disruption of membranes.39 Considering the strongly lipophilic character of curcumin and its bilayer localization, we suggest that the collision mechanism prevails in the mode of action of CFN. Since this mechanism is not associated with membrane disruption, it may allow longer functional durability of the CFN-coated surfaces. To the best of our knowledge, this is the first report of curcumin-functionalized active antimicrobial surfaces. Obviously, physical contact of these surfaces with microbial cells was necessary for realization of their bactericidal potential. Inclusion of glucose in the interface layer of the vesicles facilitated their binding with bacteria based on ligand−receptor interaction.29 2563

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of intermediate and final products formed during synthesis of diacetylene monomers as shown in Figure 2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +972-3-9683609; Fax +972-3-9683622; E-mail: vrodov@ agri.gov.il. Present Address

⊥ Navneet Dogra: Department of Chemical & Environmental Engineering, School of Engineering & Applied Science, Yale University, 55 Prospect Street, New Haven, Connecticut 06511, United States.

Funding

This research was supported by the US-Israel Binational Agricultural Research and Development (BARD) Grants US4471-11F and US-4680-13C. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No 705/14. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to David P. Clark, Department of Microbiology at Southern Illinois University in Carbondale, for providing stock cultures of E. coli and B. cereus.

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ABBREVIATIONS USED AAUC, added area under the curve; AFM, atomic force microscopy; CFN, curcumin-functionalized nanovesicles; CFU, colony-forming units; DAPI, 4′,6-diamidino-2-phenylindole; DCM, dichloromethane; DMPC, 1,2-dimyristoyl-sn-glycero-3phosphocholine; IC50, half-maximal inhibitory concentration; MBCD, methyl-β-cyclodextrin; MIC, minimum inhibitory concentration; NHS, N-hydroxysuccinimide; NYDB, nutrient yeast dextrose broth; PBS, phosphate buffer saline; PDA, polydiacetylene; TEM, transmission electron microscopy; TSA, tryptic soy agar; TSB, tryptic soy broth

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