Sonochemically Processed Cationic Nanocapsules - American

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Sonochemically Processed Cationic Nanocapsules: Efficient Antimicrobials with Membrane Disturbing Capacity Margarida M. Fernandes,† Antonio Francesko,† Juan Torrent-Burgués,† F. Javier Carrión-Fité,‡ Thomas Heinze,§ and Tzanko Tzanov*,† †

Grup de Biotecnologia Molecular i Industrial, Department d’Enginyeria Química, Universitat Politècnica de Catalunya, Rambla Sant Nebridi 22, 08222 Terrassa, Spain ‡ Instituto de Investigación Textil y C.I. de Terrassa Laboratorio de Tensioactivos y Detergencia, Departamento de Ingeniería Textil y Papelera, Universitat Politècnica de Catalunya, Colom 1508222 Terrassa, Spain § Center of Excellence for Polysaccharide Research, Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich Schiller University of Jena, Humboldtstraße 10, 07743 Jena, Germany ABSTRACT: Bacterial-mediated diseases are a major healthcare concern worldwide due to the rapid spread of antibioticresistant bacteria. One strategy to manage the bacterial infections while avoiding the emergence of resistant strains implies specific targeting and disruption of bacteria membranes. This work evaluates the potential of nanostructured biopolymer derivatives, nanocapsules (NCs), to disrupt the bacteria cell walls and effectively kill planktonic microorganisms. Two biopolymers, chitosan and cellulose, were chemically modified to synthesize derivatives with improved cationic character (thiolated chitosan and aminocellulose) prior to their processing into nanocapsules via a one-step sonochemical process. The interactions of NCs, displaying an average size of around 250 nm, with bacteria membrane were evaluated using two membrane models: Langmuir monolayers and liposome bilayers composed of a L-α-phosphatidylglycerol phospholipid extracted from Escherichia coli. NCs possessed improved membrane disturbing capacity in comparison to the nonprocessed biopolymer derivatives, by drastically increasing the monolayer fluidity and inducing more than 50% leakage of a dye inserted in the bilayered liposomes. In addition, membrane disturbance was directly proportional to the NCs cationic charge. Whereas evidence showed that thiolated chitosan and aminocellulose interacted with the bacteria membrane through a “carpet model”, the NCs were found to induce larger surface defects and high local perturbance through a “detergent model”. Importantly, the degree of disruption caused by the biopolymer derivatives and NCs correlated well with the antimicrobial capacity against Escherichia coli, selectively killing bacteria cells without imparting toxicity to human fibroblasts.

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assess their toxicity toward both human and bacterial cells.7−9 The lipid bilayer of a cell membrane comprises two weakly coupled monolayers that serve as a continuous barrier between the cellular content and the local environment. Therefore, both monolayers and liposomes are considered critical model interfaces to study nanobiointeractions.8,10 One widely accepted mechanism is that nanoparticles interact preferentially with microbial membranes through electrostatic interactions causing an increase in the cell membrane permeability, osmotic damage, and flow of cytoplasmic components out of the cell, eventually leading to cell death.3 Such mechanism reduces the possibility of developing new resistant strains because bacteria membrane is highly evolutionarily conserved and therefore unlikely to be changed by a single gene mutation.11−14 In addition, nanoparticles are also able to tackle multiple biological pathways

INTRODUCTION The antibiotics discovery in the early 20th century disclosed a new era in the treatment of microbial infections. They saved countless lives, extended life expectancies, and permitted previously deadly medical procedures such as surgeries.1 However, their widespread use has accelerated the emergence of antibiotic-resistant bacteria and nowadays infectious diseases are considered one of the greatest health challenges.2 There is an urgent need for new antimicrobial agents with low susceptibility to resistance development. Active molecules processed into nanosized particles are largely claimed to be more efficient antibacterial agents than their nonprocessed counterparts.3−5 The high surface area to volume ratio and unique physicochemical properties are believed to contribute to their high antimicrobial capacities even in low counts.6 The exact mechanisms by which nanoparticles interact with bacteria are not fully understood. Nevertheless, their interaction with cell membrane models such as phospholipid monolayers (Langmuir monolayers) and liposomal bilayers have been increasingly studied to indirectly © 2014 American Chemical Society

Received: December 22, 2013 Revised: March 14, 2014 Published: March 18, 2014 1365

dx.doi.org/10.1021/bm4018947 | Biomacromolecules 2014, 15, 1365−1374

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Figure 1. Chemical Structure of predominant species of E. coli extract L-α-phosphatidylglycerol (sodium salt) phospholipid. chitosan derivative. Microcrystalline cellulose (Fluka, Avicel PH-101) dried at 105 °C for 2 h was used for the preparation of the 6-deoxy-6(ω-aminoethyl) aminocellulose derivative. E. coli extract L-αphosphatidylglycerol (sodium salt; E. coli PG, average Mw = 761 g/ mol) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL; Figure 1). 1,2-Iminothiolane HCl, 5,5′ dithiobis(2-nitrobenzoic acid) (Ellman’s reagent), 1,6-diphenyl-1,3,5-hexatriene (DPH), 2-nitrophenyl β-D-galactopyranoside (ONPG), and 2′,7′-dichlorofluorescin diacetate (DCF-DA) were purchased from Sigma-Aldrich (Spain). AlamarBlue Cell Viability Reagent was purchased from Invitrogen, Life Technologies Corporation (Spain). Human foreskin fibroblasts cell line BJ-5ta (ATCC-CRL-4001) and the bacteria strain Escherichia coli (E. coli, ATCC 25922) were purchased from American Type Culture Collection (LGC Standards S.L.U, Spain). Ultrapure water (18 MΩ cm) was obtained with a Simplicity UV Millipore equipment and used throughout the work. All other chemicals were obtained from SigmaAldrich (Spain) and used without further purification. Biopolymer Functionalization. Chitosan Thiolation and Characterization. The synthesis of TC was carried out in a onestep coupling reaction between 2-iminothiolane HCl (Traut’s reagent) and primary amino groups of chitosan.20 Briefly, chitosan was dissolved in CH3COOH (1%, v/v) to reach 1% solution (w/v). The pH of the solution was adjusted to 6 with NaOH (5 M) and Traut’s reagent added to reach the chitosan/Traut’s reagent ratio of 5:2 (w/ w). The mixture was stirred overnight at closed vessel in dark and thereafter dialyzed two times for 24 h against 1 mM HCl containing NaCl (1%) and finally for three days in 1 mM HCl. The dialyzed thiolated derivative (TC) was freeze-dried and stored at +4 °C until further use. The amount of reduced thiol groups was determined spectrophotometrically using Ellman’s reagent.21 Cellulose Amination. Tosyl cellulose was synthesized according to the procedure described by Rahn et al.22 Cellulose was dissolved in N,N-dimethylacetamide/LiCl (DMA/LiCl) and allowed to react with p-tosyl chloride (TosCl) in the presence of triethylamine (Et3N) at 8− 10 °C. The degree of substitution of the tosylate groups (DSTos) was adjusted by the application of different molar ratios of p-tosyl chloride and triethylamine. A total of 20 equiv of the ethylendiamine were added, and the temperature of the reaction mixture was increased to 100 °C and stirred for 3 h. The product was isolated by precipitation in 200 mL of water. The precipitate was filtered off and washed four times with 150 mL of isopropanol and four times with 150 mL water. The product was dried in vacuum at 40 °C. Sonochemical Preparation of Nanocapsules. TC and AC nanocapsules (NCs) were prepared by an adaptation of Suslick method.23 Before sonication, the pH of the AC and TC aqueous solution was adjusted to 6 using 1 M HCl and 1 M NaOH, respectively. Briefly, a two-phase solution containing 70% of 1 g/L biopolymer derivatives aqueous solution and 30% of commercial sunflower oil (organic phase) was prepared and placed into a thermostatted (8 °C ± 1 °C) sonicator cell. The NCs were synthesized with a high-intensity Vibra-Cell VCX 750 ultrasonic processor (Sonics and Materials, Inc., U.S.A.) using 20 kHz Ti horn at 35% amplitude. The bottom of the probe was positioned at the aqueous−organic interface, employing an acoustic power of ∼0.5 W/ cm3 for 3 min using an ice-cooling bath to maintain the low temperature. The resulted suspension was kept at 4 °C for 24 h and the nonreacted organic solvent was removed by three washing cycles with water and centrifugation at 800 rpm for 15 min. Nanocapsules Characterization. The NCs size was measured by DLS using DL135 Particle Size Analyzer (Cordouan Technologies, France). Three samples of each nanoparticle suspension were measured at room temperature, acquiring five measurement cycles

found in broad spectrum of microbes and many concurrent mutations would have to occur in bacteria in order to develop resistance.3 There have been evidence that cationic properties of nanoparticles are the main driving force for the disruption of both monolayer and bilayer models. Positively charged polymers have been reported to efficiently create holes in lipid layers. In general, the high surface charge density in polycations (particularly with ζ-potential value above +40 mV) and chain mobility are important parameters for achieving strong bactericidal effect.15 Conversely, particles with lower positive and opposite surface charges do not initiate holes but are rather limited to expand the already existing defects, and thus display lower membrane disturbing capacities.16,17 Indeed, engineering a positive surface charge to bind electrostatically to anionic bacteria membrane is the most widely used approach to achieve active bacteria targeting. Under physiological conditions, bacteria cell walls are negatively charged due to the presence of lipoteichoic acids, peptidoglycan layers, and phospholipids, making this approach comprehensive and suitable even for polymicrobial infections. However, positive surfaces may also impart toxicity toward human cells, especially when used in inappropriate concentrations, due to a lack in specificity. This work intends to combine the engineering of highly cationic biopolymer derivatives with their processing into nanoscale structures as an integrated strategy for the development of biocompatible and efficient antibacterial agents. To this end, chitosan and cellulose were chemically modified to obtain thiolated chitosan (TC) and aminocellulose (AC), derivatives with improved cationic character, prior to their processing into nanocapsules via application of high intensity ultrasound. This novel, fast and one-step sonochemical synthesis develops physiologically stable biopolymeric core−shell nanocapsules.18 TC was obtained by the direct coupling of chitosan with 2iminothiolane HCl into a highly cationic derivative due to the presence of amidinium cation. AC was obtained using a twostep modification approach: tosylation of cellulose and subsequent nucleophilic displacement reaction with ethylendiamine to obtain the AC derivative with protein-like selfaggregation behavior.19 Despite several reports on the synthesis of TC and AC nanostructures using other than sonochemical methods, their antimicrobial properties and mechanisms of action have never been claimed. Considering the importance of the bacterial cell membrane disruption as an antibacterial strategy to avoid the resistance development, the interaction of these NCs with Langmuir monolayers and liposome bilayers comprising the Escherichia coli (E. coli) phospholipid L-α-phosphatidylglycerol (PG) was investigated and correlated with in vitro antibacterial assays against planktonic bacteria.

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EXPERIMENTAL SECTION

Materials. Medical grade chitosan (∼15 kDa, DDA 87%) obtained from Kitozyme (Belgium) was used for the preparation of thiolated 1366


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biopolymer derivative solution and NCs (100 μg/mL) at a ratio (v/v) of 1:1. NCs, and biopolymer derivatives, induced DPH release as a result of dye leaking from LUVs membrane was monitored fluorimetrically (excitation at λ = 350, emission at λ = 425 nm) for 45 min at 37 °C on a TECAN Infinite M200 plate reader. The timedependent leakage as the normalized fraction of released DPH is given by

with 1% signal-to-noise ratio. The data were analyzed using NanoQ 1.2.1.1 software to calculate the mean particle diameter. The determination of zeta potential was carried out using a Zetasizer Nano Series (Malvern Instruments Inc., Worcester, U.K.) after appropriate dilution of NCs using ultrapure-grade water at pH 6. The shape and morphology of NCs was visualized with a scanning electron microscope (SEM) JEOL JSM-7100F and a Cryo-SEM using a JEOL JSM-6510 Field Emission Scanning Electron Microscope equipped with a Gatan ALTO 1000 cryo-preparation system (Gatan Inc., Abingdon, U.K.). SEM samples were prepared by applying a small drop of NCs suspension in a glass slide and allowing it to dry at room temperature before gold−palladium sputtering. Cryo-SEM samples were frozen with liquid nitrogen in a preparation chamber at −240 °C, sputter-coated with gold−palladium, and examined in a frozen state at 10 kV and −190 °C. Interaction of Nanocapsules with Cell Membrane Models. Langmuir Monolayer Measurements. TC and AC stock solutions and TC and AC NCs suspensions at 1 mg/mL, pH 6.0 were used. The samples were then diluted 500 times with Milli-Q water (18 MΩ cm) to a concentration of 2 μg/mL and studied in the subphase for their interaction with a E. coli PG monolayer. The measurements were performed with a Langmuir trough (KSV NIMA Langmuir−Blodgett Deposition Troughs, Model KN2002, Finland) with a total area of 273 cm2 mounted on an antivibration table and housed in an insulation box. The trough was filled with a subphase solution that was either Milli-Q water (control) or the TC and AC diluted solutions and NCs suspension, and 20 μL of a 0.5 mg/mL phospholipid solution in HPLC grade chloroform was carefully spread onto the subphase with a gastight syringe (50 μL). After chloroform evaporation (10 min), the floating film was continuously compressed at the air−water interface at a linear speed of 25 mm/min (10.2 Å2 molecule−1 min−1). The temperature of the system (22 ± 1 °C) and the pH of the biopolymer derivatives (pH 6.0) and NCs were maintained constant, because these parameters could affect the phase behavior of the monolayer, altering significantly the shape and location of the isotherms. The surface pressure (π) was recorded against the area (A), measured with a Wilhelmy balance plate (Pt) connected to an electrobalance. The isotherms provide information about the film states, phases, and phase transitions, where π is the difference between the surface tension of pure water (γ0) and that of the film (γ), that is, π = γ0 − γ.24,25 The compression modulus (in-plane elasticity) Cs−1 was calculated from the slope of the π versus A isotherm and discloses important information about the phase transitions occurred in the monolayer:26 ⎛ dπ ⎞ Cs−1 = − A⎜ ⎟ ⎝ dA ⎠T

L(t ) =

I(t ) − I0 Imax − I0

where I(t) is the time-dependent fluorescence intensity, I0 is the initial intensity before the addition of biopolymer derivatives and NCs, and Imax is the maximum fluorescence intensity upon the complete leakage of the dye, which was induced by the addition of 10% (v/v) Triton X100 solution. Nanocapsules Antimicrobial Activity. A gram-negative Escherichia coli (E. coli ATCC 25922) obtained from ATCC collection was used in all antimicrobial assays. For the preparation of bacterial inoculum, a single colony from the corresponding stock bacterial cultures was used and allowed to grow overnight at 37 °C and 230 rpm in Mueller Hinton (MH) broth. Bacteriostatic Activity Assay. Bacterial growth inhibition of biopolymer derivatives and NCs suspension were performed against E. coli by using a turbidity-based microdilution assay in MH broth.28 Briefly, different concentration of working solutions of the biopolymer derivatives and NCs (1−100 μg/mL) were prepared by serial dilutions of the stock solutions (1000 μg/mL) in MH. A 96-well polypropylene microplate (Corning #3359) well was filled with 75 μL of biopolymer derivatives and NCs and 75 μL of bacteria inoculum to a final concentration of bacteria of ∼5 × 105 CFU/mL. The growth of bacteria was monitored by recording the optical density at 600 nm (corrected for the background absorbance of each blank) every 10 min during 18 h using a microplate reader (Infinite M200, Tecan, Austria). The specific growth rates (min−1) were obtained from the exponential phases of the individual growth curves and related to the specific growth rate of the negative control. The reported inhibition growth rates are mean values of five independent experiments with standard deviation as a source of error. Bactericidal Activity Assay. The bactericidal activity of TC and AC and NCs suspension was evaluated by assessing the bacterial viability through the measurement of the reduction of the nonfluorescent dye AlamarBlue by bacterial metabolites. The overnight cultures were harvested by centrifugation and washed twice with a 0.9% solution of NaCl at pH 6.5. The inoculated bacterial culture was diluted with sterile saline solution (0.9% NaCl, pH 6.5) until reaching the solution absorbance of 0.28 ± 0.1 at 600 nm, which corresponds to 1.5−3.0 × 108 CFU/mL. Thereafter, 200 μL of cell suspension was added to 1800 μL of different concentrations of biopolymer derivatives and NCs in a 24-well plate at 37 °C and 100 rpm for 2 h. After 2 h, samples from each well were collected and 10 μL of each sample were transferred to a 96-well polypropylene microplate (Corning #3359) previously filled with 100 μL of an AlamarBlue solution (10% v/v in MH broth). The plate was further incubated for 6 h, at 37 °C in the dark, and the absorbance at 570 nm was measured, using 600 nm as a reference wavelength. Bacterial cell metabolic activity (%), indicating the percentage of live bacteria, was determined for each concentration and compared with that of cells incubated only with live bacteria. Data are expressed as the mean of three measurements, with a standard deviation as a source of error. Membrane Permeabilization Assay. The extent of NC-induced membrane permeabilization was determined by measuring the release of cytoplasmic β-galactosidase activity from E. coli into the culture medium using ONPG as the substrate.29 Briefly, E. coli cultures were harvested, washed, and resuspended in 0.9% NaCl solution. The final cell suspension was adjusted to obtain a A420 of 1. Biopolymer derivatives and NCs (100 μL) were mixed with bacteria suspension (100 μL) in a 96-well plate and 30 mM ONPG acetone solution (10 μL). The production of ο-nitrophenol over time was monitored by the increase in A420 using a spectrophotometer.

(1)

while the minimal value in the plot of Cs−1 versus π indicates a phase transition the maximum allows the classification of these transitions as liquid-expanded (12.5 < Cs−1 < 100 mN/m), liquid-condensed (100 < Cs−1 < 250 mN/m), and condensed (Cs−1 > 250 mN/m). Liposomes Bilayer Measurement. Large unilamellar vesicles (LUVs) of E. coli PG loaded with the fluorescent probe 1,6diphenylhexatriene (DPH) were prepared according to the method previously described.27 Briefly, LUVs with a fluorophore/lipid molar ratio of 1:100 were prepared by dissolving 7.4 mg of E. coli PG in a chloroform/methanol (5:1) solution containing DPH dye (0.023 mg/ mL) and mixed very well. The evaporation of the solvents was performed in a rotary evaporator (100 rpm connected to a vacuum pump) for at least 2 h until a thin film was formed. Exposing the film under nitrogen atmosphere for 1h ensured complete evaporation. The dried films were then hydrated with a Tris/NaCl buffer 10:100 mM (pH 7.4) and resuspended from the walls of the glass tube by vigorous vortexing for 10 min followed by vigorous shaking (200 rpm) on the rotary evaporator (without vacuum) at 45 °C for 1 h. After hydration, the lipid solution was subjected to three freeze−thaw cycles, by freezing in ice and thawing at 50 °C. Large unilamellar liposomes (LUVs) were generated by 10 extrusions through polycarbonate filters (pore size: 1 μm). To monitor the bilayer permeabilizing activity of NCs, LUVs loaded with the fluorescent probe DPH were mixed with 1367


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Figure 2. Synthesis pathways for (A) chitosan thiolation and (B) cellulose amination.

Figure 3. Cryo-SEM and SEM micrographs of nanocapsules suspension. (A) Cryo-protected aminocellulose nanocapsules; (B) cryo-protected thiolated chitosan nanocapsules; (C) aminocellulose nanocapsule; (D) thiolated chitosan nanocapsule. Cytotoxicity Evaluation. Cell Culture. To determine the potential toxicity of the NCs, BJ-5ta cells at passage 11 were used. The cells were maintained in four parts Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4 mM L-glutamine, 4500 mg/L glucose, 1500 mg/L sodium bicarbonate, 1 mM sodium pyruvate, and one part of Medium 199, supplemented with 10% (v/v) of fetal bovine serum (FBS) and 10 g/mL hygromycin B at 37 °C in a humidified atmosphere with 5% CO2, according to the recommendations of the manufacturer. The culture medium was replaced every 2 days. At preconfluence, cells were harvested using trypsin-EDTA (ATCC-30− 2101, 0.25% (w/v) trypsin/0.53 mM EDTA solution in Hank’s BSS without calcium or magnesium). AlamarBlue Assay. Cells were seeded at a density of 4.5 × 104 cells/well on a 96-well tissue-culture-treated polystyrene plate (Nunc) the day before experiments and then exposed to different concentrations of NCs (1000, 100, and 1 μg/mL diluted in DMEM) at a final volume of 150 μL and incubated at 37 °C in a humidified atmosphere with 5% CO2. After 24 h contact with cells, the NCs were removed and the cells were washed twice with PBS and stained for signs of toxicity using AlamarBlue assay. Briefly, 100 μL of 10% (v/v) AlamarBlue reagent in DMEM was added to the cells and incubated for 4 h at 37 °C, after which the absorbance at 570 nm was measured, using 600 nm as a reference wavelength, in a microplate reader (Infinite M 200 plate reader, Tecan). The quantity of resorufin formed is directly proportional to the number of viable cells. BJ5ta cells relative viability (%) was determined for each concentration of

NCs and compared with that of cells incubated only with cell culture medium. H2O2, 500 μM, was used as a positive control of cell death. Data are expressed as the mean of three measurements, with a standard deviation as a source of error.

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RESULTS AND DISCUSSION Biopolymer Functionalization. Biopolymers such as cellulose and chitosan are increasingly researched for pharmaceutical and biomedical applications due to their biodegradability, biocompatibility, and large-scale availability.30−34 Despite these properties, their modification has been widely proposed to improve their functional properties. In this work, chitosan and cellulose were chemically modified with the purpose of increasing their cationic properties as a way of imparting improved antimicrobial activities. Chitosan was thiolated by direct coupling with 2iminothiolane HCl (Figure 2A), a well-established method for thiolation of proteins35 and since recently polysaccharides.36 The modification of chitosan originated a biopolymer derivative with free thiol functionalities, determined spectrophotometrically using Ellman’s reagent (515 ± 53 μmol/g), and high positive charge (+ 29 ± 1 mV measured by ζ-potential) due to the established amidine linkages.37 We have previously demonstrated that the thiolation with 2-iminothiolane HCl 1368


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The mean hydrodynamic diameters, calculated based on number-average size from photon correlation spectroscopy measurements were similar among the two NCs: 268 ± 7 nm for AC and 247 ± 4 nm for TC, which was also confirmed by SEM images (Figure 3 C and D). The capsules were developed without the addition of surfactants, a tensioactive agent commonly used to improve the stability of nano/microspheres suspensions.44 Previous reports on sonochemically generated nanoscaled chitosan have shown the need to add a surfactant during the process of capsules generation in order to stabilize and diminish the size and polydispersity of the obtained capsules.40 In this work, no surfactant was applied to obtain small and physically stable capsules, which is believed to be due to the polyelectrolyte nature of aminocellulose and thiolated chitosan. Indeed, the NCs were found to be extremely stable, as indicated by their high ζ-potential values of +103 ± 2 mV for AC NCs and +81 ± 1 mV for TC NCs. ζ-Potential is a measure of the magnitude of electrostatic repulsion/attraction between particles, and values above ±60 mV are an indication of high particle stability. The ζ-potential is also defined as the electrical potential at the surface of the hydrodynamic shear around the colloidal particle and, thus, regarded as a characteristic parameter for the particle charge.45 The positive ζ-potential value measured for the herein obtained NCs indicates that these are cationic particles possessing the intended key characteristic for efficient disruption of lipid bilayers.7 Interaction of Nanocapsules with Bacterial Cell Membranes. Interaction with Langmuir Monolayers. The Langmuir film balance technique measures the surface pressure (π) as a function of the available area (A) for each phospholipid molecule in the monolayer in the course of compression. The resulting isotherms (π−A) demonstrate the monolayer phase behavior when the molecular packing gradually increases, which is an indirect measure of the orientation of the phospholipid molecules on the air−aqueous interface.24,25 In a general situation, when the phospholipid is spread on the air−water interface at large molecular areas, the monolayers exist in a gaseous phase (G). After compression, it evolves to a liquidexpanded phase (LE), followed by a liquid-condensed phase (LC), finally reaching the condensed phase (C) at high molecular densities. This technique has been used to elucidate the interactions of several antimicrobial agents with cell membrane models at the molecular level,38,46−50 thus, indirectly assessing its antimicrobial potential.

improved the antimicrobial capacity of chitosan through a better disruption of the cell membrane wall due to its improved cationic character.38 The AC, on the other hand, was synthesized by applying chemo- and regioselective nucleophilic displacement reaction of p-toluenesulfonic acid ester of cellulose with ethylendiamine (Figure 2B),33,39 resulting in a derivative with a high positive charge (+ 37 ± 2 mV). Nanocapsules Preparation and Characterization. TC and AC NCs were developed by the sonochemical method first reported by Suslick and Grinstaff23 and recently used to synthetize biopolymer- and protein-based nano/microcapsules.40−42 The NCs were obtained from aqueous solutions of TC and AC using vegetable oil to complete the two-phase mixture. This method uses an ultrasonic emulsification process in a biphasic system to synthesize capsules dispersed in the aqueous solution. The synthesis is assisted by effective intermolecular interactions (hydrogen bonding, van der Waals, hydrophobic, and electrostatic interactions) that occur during the emulsification process in which the biopolymer molecules, initially present in the aqueous phase, localize at the interface of the droplet, thus, generating a capsule with a biopolymeric shell and an oil core.23,43 Spherical AC and TC NCs were successfully generated by this method, as observed by scanning electron microscopy images (Figure 3), similarly to biopolymer-based microspheres (chitosan) previously reported by Skirtenko and co-workers.18 The NCs also demonstrated narrow size distributions, as revealed by both cryo-SEM images, where bigger particles depicted are oil bubbles (Figure 3A,B), and dynamic light scattering (DLS) results (Figure 4).

Figure 4. Thiolated chitosan and aminocellulose NC size distributions.

Figure 5. Surface pressure−area isotherm (π−A) of E. coli PG monolayer on water and on 2 μg/mL of aqueous solution of biopolymer derivatives and nanocapsules (A) and respective compression modulus (B). 1369


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isotherms (A = 230 Å2 for AC NCs and 200 Å2 for TC NCs). Interestingly, the isotherms displayed a well-defined plateau at a surface pressure of around 12−15 mN/m (Figure 5A). This plateau suggested that a reorientation or reorganization of E. coli PG molecules within the LE state (Cs−1 ≈ 55 mN/m) occurred, indicating an effective loosening of the monolayer by the presence of the NCs. These maintain their position at the interface and keep a constant interaction with the E. coli PG heads and tails (Scheme 1B, small molecular areas) until reaching high surface pressures values, close to the collapse of the monolayer. The drastic decrease on the value of Cs,max−1 of phospholipid monolayer, reflected an increase in the fluidity and compressibility of monolayers as a consequence of the NCs disturbing effect, indicative of their improved ability to interact and disrupt the membrane. This peculiar behavior could be related to the improved cationic properties of the capsules (high ζ-potential) and the higher surface area available for interaction with the monolayer, inducing stronger steric effect and thus disturbing/disruption capacity. Being nanosized and spherical-shaped allow them for even more efficient interactions with PG heads and tails (Scheme 1B, large molecular areas).52 In fact, the disturbance of the monolayer at large areas per molecule seems to be charged dependent, that is, the higher the particle surface charge the bigger the isotherms expansion. AC NCs possessed higher ζ-potential (103 mV) than TC NCs (81 mV), inducing a higher degree of disturbance in the monolayer. These changes in the isotherms provided a clear indication that both biopolymer derivatives and NCs, thereof possess membranedisturbing activity, being the effect stronger with NCs, an important feature to avoid drug resistance development. To give better insights of the possible antimicrobial mechanism of action of both biopolymer derivatives and NCs, the values of the compression modulus of the E. coli PG monolayers at a surface pressure of 30 mN/m were also analyzed (Table 1). This surface pressure is believed to

Prior to testing the biopolymer derivatives and their respective NCs with the model cell membrane, their surface activity in the absence of a phospholipid was tested. None of the solutions were surface-active as no change in the surface pressure upon barrier compression was indicated (results not shown). The isotherm for the E. coli PG with only water in the subphase (Figure 5A) presented a lift-off at molecular areas of around 80 Å2, experiencing after that a phase transition from G to LE state, represented by a maximum in the compression modulus Cs,max−1 around 100 mN/m (Figure 5). The inability of reaching higher values of compression modulus (Cs−1) is due to the fact that the E. coli PG phospholipid is a nonpure compound, presenting a mixture of structurally different PG phospholipids. Namely, the presence of cyclopropane in the alkyl chain of the predominant phospholipid (Figure 1) is believed to increase the monolayer fluidity, thus, lowering the compression modulus values and impeding the monolayer to organize into a more packed structure. The biopolymer derivatives in the subphase presented similar isotherms, lifting off at slightly bigger molecular areas (A = 100 Å2 for AC and 115 Å2 for TC) and reaching similar maximum compression modulus (Cs,max−1 = 96 mN/m for AC and 91 mN/m for TC). These values revealed a phase transition from G to LE state, similarly to what happened with the control. The monolayer expansion indicated that biopolymer derivatives were able to disturb the phospholipid monolayer at large molecular areas, owing to their cationic character, interacting through electrostatic interactions with the negatively charged phospholipid polar head and through hydrophobic interactions with the tails at the interface, as a consequence of penetration of the biopolymer derivatives among the phospholipid chains (Scheme 1A, large molecular areas). This mechanism of action Scheme 1. Model for the Interaction of the Biopolymer Derivatives (A) and Nanocapsules (B) with the Phospholipid Monolayer

Table 1. Compression Modulus (Maximum and at a Surface Pressure of 30 mN/m) of E. coli PG Monolayers with Water, Biopolymer Derivatives, or Nanocapsules in the Subphase E. coli PG Cs,30−1

sample control (H2O) AC AC NCs TC TC NCs

toward the membrane was previously reported to rule the chitosan and TC interaction with monolayers of its structurally related phospholipid DPPG.38,49,51 However, at small molecular areas the Cs,max−1 is similar to the control which indicates that the high surface density of the monolayer (small molecular areas) may favor the expelling of biopolymer moieties into the subphase to some extent. Nevertheless, the isotherms were shifted toward large molecular areas suggesting that the biopolymer derivatives are still interacting with the monolayer (Scheme 1A, small molecular areas), as previously suggested by other studies focusing on chitosan DPPG monolayer disturbing capacity.49−51 Further processing the biopolymers into NCs increased this membrane-disturbing capacity even at very low concentrations (2 μg/mL), by greatly increasing the lift-off area of the

(mN/m) 100 96 46 91 38

Cs,max−1 (mN/m) 109 97 62 94 56

correspond to the lateral pressure of naturally occurring cell membranes,53 so that the increase of the fluidity of the membrane may predict the ability of these compounds to effectively disturb the cell membrane. The NCs were able to drastically lower the compression modulus of the E. coli PG monolayer when compared to the control (Table 1). On the other hand, AC and TC induced little effect on the compression modulus at a surface pressure of 30 mN/m, possibly due to the low concentration tested (2 μg/mL). Interaction with Vesicles Bilayer Model. A diphenylhexatriene (DPH) leakage assay from E. coli PG liposomes with a mean-average size of 1 μm was evaluated to probe the effects of the biopolymer derivatives and NCs on cell membranes. Leakage of DPH from the liposomes provides information 1370


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isotherms toward higher molecular areas. These observations suggest that the NCs interact more efficiently with the membrane wall, being able to cause more defects than the derivatives in solution. The NCs mechanism of action to disturb the bacterial cell membrane might be through the “detergent model”, inducing large surface defects/holes as a result of the strong electrostatic interactions and thus improved steric effect occurring at the interface of the cell.56 The biopolymer derivatives probably act through the “carpet model” forming a film on the surface of the membrane, also through electrostatic interactions with the negatively charged polar heads, and through interaction with phospholipid hydrophobic tails, therefore, expanding the existing defects.11 Assessment of the Nanocapsules Antimicrobial Activity. Three different antimicrobial assays were performed to corroborate the findings for disruption of cell membrane models. First, the E. coli inner membrane (IM) permeabilization was evaluated as a function of cytoplasmic β-galactosidase release. When E. coli inoculum was incubated with the NCs, an immediate release of β-galactosidase was observed (Figure 7).

about the bilayer integrity since this dye is inserted in the liposome bilayer oriented parallel to the lipid acyl chain axis. Figure 6 shows the time-dependent DPH leakage, L(t), for

Figure 6. Time-dependency study of diphenylhexatriene (DPH) dye leakage from large multilamellar vesicles (MLVs) and percentage of liposome disruption in relation to Triton X-100.

Milli-Q water, biopolymerderivatives, NCs, and Triton X-100, a surfactant that induces complete disruption of the liposome structures, and thus the leakage of the dye. The TC and AC did not induce leakage of the dye, indicating inability (at the tested concentrations of 100 μg/mL) to disrupt this model membrane. The interaction of antimicrobial agents with bacterial cell membranes represent a multiple step process that includes an initial recognition, bacteria attachment, insertion into the membrane, and finally disruption of the cell wall.54 It is believed that biopolymer derivatives might be attached to the lipid bilayer without inducing major defects at 100 μg/mL. Both TC and AC NCs presented different tendencies. TC NCs induced a rapid leakage of the dye causing 57% of liposomes disruption after 30 min, after which reached a steady state. This behavior suggested that TC NCs remain on the bilayer surface instead of attaching to other liposomes; otherwise, a continued increase in the leakage until all the dye is released from all liposome would be observed.55 AC NCs, on the other hand, induced the dye leakage with a 15 min delay, after which a continuous fluorescence increase was observed. After 45 min, 26% of the liposomes were disrupted, reaching 100% after 2.5 h of contact with the liposomes (results not shown). The initial attachment of AC NCs to bacteria could explain the delay on their action.54 Despite the slower kinetics, the AC NCs induced complete leakage of the dye, which suggests their improved ability to cause bacterial membrane disruption. The higher cationic surface charge might be responsible for this difference, similar to the previously explained Langmuir monolayer measurements. Determining the physical disruption mechanism by which the NCs mediate monolayer expansion and liposome leakage is important to gain insight into how NCs may interact with bacteria cell membrane. NCs with particle sizes of around 250 nm in very low concentrations (2 μg/mL in Langmuir and 100 μg/mL in liposomes assay), were able to shift the Langmuir isotherms toward higher molecular areas, drastically increasing the monolayer fluidity at a surface pressure typically found in cell membranes, induced the appearance of new phase transition and caused dye leakage from liposomes. On the other hand, the nonprocessed derivatives (at the same concentrations as the NCs) were only effective in shifting the

Figure 7. Release of cytoplasmic β-galactosidase activity from the E. coli cells in contact with the biopolymer derivatives at 100 μg/mL, corresponding nanocapsules and PBS (control).

The biopolymer derivatives in solution did not induce the enzyme release, which proves the lack of efficacy in disrupting the cell membrane at a concentration of 100 μg/mL (same concentration used in DPH-leakage from liposomes assay). The previous reports on the β-galactosidase release from E. coli in the presence of biopolymers (chitosan) in solution showed an effective IM disruption capacity.57 However, this was evaluated with higher biopolymer concentration (10 mg/mL) than the one used in this study. The metabolic activity of E. coli was further measured in the presence of NCs and cationic biopolymer derivatives at increasing concentrations using AlamarBlue reagent. This cell viability reagent is used to assess the metabolic activity of mammalian or bacterial cells in culture media, providing information about the percentage of live cells.58 The AC in solution inhibited 20% of E. coli metabolic activity, which decreased by more than 40% after NCs processing (Figure 8A). In addition, the cell metabolic activity decreased in a concentration-dependent manner with AC NCs suspension. Whereas TC in solution did not induce any inhibition on the E. coli metabolic activity, the TC NCs suspension reached 35% of inhibition. This assay provided the proof that the NCs possess improved antibacterial activity against E. coli in comparison to 1371


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Figure 8. Effect of the nanocapsules and biopolymer derivatives at different concentrations (0−100 μg/mL) in the reduction of metabolic activity of E. coli using AlamarBlue (A) and on the inhibition of E. coli growth.

than 95% viability reduction (Figure 6). The TC was less toxic, inducing up to 40% reduction. When concentration was decreased to the levels that showed high antibacterial potential (100 μg/mL), the TC did not induce any toxicity to fibroblasts. Interestingly, at this concentration the AC NPs did not induce toxicity whereas the AC in solution induced 80% viability reduction. Previous studies have shown that amine-containing polymers induce mammalian cell toxicity due to their positive charges,16,60 which could explain the high toxicity observed for AC in solution at this concentration. The reason why NCs are not toxic at this concentration might be related with less availability of amino groups in the form of capsules in suspension than in solution, despite the NCs higher surface charge observed in this study. The amino groups in AC are thought to be partially involved in the particle formation and stabilization, which would explain the decrease in toxicity. Previous studies have shown that surface charge plays an important role on the selectivity to target only bacterial membranes. The selective targeting appears to be a result of the highly cationic surface charge (above +47 mV) interacting with the more negatively charged microbial membranes when compared to human cell membrane.61 The NCs used in this study possess a higher cationic surface charge, which might explain the nontoxicity of TC and AC NCs in comparison to AC in solution at bactericidal effective low concentrations.

the starting biopolymer derivatives. AC NCs were more bactericidal than TC NCs, which is in good agreement with the results from both Langmuir monolayer and DPH-leakage assay. Finally, the bacteriostatic activity was also evaluated and similarly to the metabolic activity results, AC NCs and the AC in solution showed better activity against E. coli, compared to TC, reaching 100% inhibition when used at a concentration of 100 μg/mL (Figure 8B). Interestingly, no significant difference was observed between the AC NCs and the AC in solution, both efficiently inhibiting bacterial growth. The high surface charge density of AC near +40 mV allows efficient interaction with the bacteria, as suggested by the DPH-leakage assay with liposomes, which is believed to allow bacteria growth inhibition but no killing.15 Nevertheless, despite that TC in solution showed lower bacteriostatic activity than AC, the activity was improved by more than 40% after NCs processing: TC NCs (100 μg/mL) inhibited more than 50% of bacteria growth, whereas the same concentration of the TC in solution inhibited only 10% of bacteria growth, corroborating the results from both Langmuir monolayer and liposomes leakage assays. The NCs studied herein showed improved antimicrobial activity by targeting more efficiently the membrane wall. The larger surface area of the NCs and improved cationic character allows them to adsorb onto the surface of the bacteria cells, inducing high local membrane perturbance.56 A combination of enhanced membrane permeability and higher local concentration of antimicrobial agent in the form of nanocapsules is thought to lead to bacteria death. In Vitro Cytotoxicity with Mammalian Cells. Cell culture based assays were used as a prescreening tool to understand the biological effects of the NCs toward human cells. The improved antibacterial activity of NCs compared to their derivatives was found to be through efficient cell membrane disruption. It was therefore important to assess whether this mechanism of action is specific only to bacterial cells or could be potentially harmful to human cells as well. The potential cytotoxicity of NCs and the starting biopolymer derivatives was evaluated using the cell viability reduction method with human fibroblasts. This method has been already employed to study the interaction of other antimicrobial conjugated electrolytes with mammalian cells.59 Both NCs and biopolymer derivatives showed high toxicity toward fibroblasts at 1000 μg/mL, being the AC the most toxic inducing more

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CONCLUSIONS The use of cationic nanostructured materials for efficient bacterial membrane disruption seems to overcome the development of microbial drug resistance, and hence is among the most promising strategies to treat infections. In this work we demonstrated the potential of sonochemically generated nanocapsules comprising cationic biopolymer derivatives, such as, thiolated chitosan and aminocellulose, to efficiently disrupt bacterial membrane and induce bactericidal activity without imparting toxicity to human cells. Two membrane models, Langmuir monolayer and liposomes bilayers, were used to study the interactions on the nano-bio interface. Evidences were provided that these cationic NCs were more efficient in membrane disintegration than the biopolymer derivatives due to the strong electrostatic interactions of NCs with bacterial membrane, inducing massive membrane surface defects, whereas the nonprocessed cationic derivatives are limited to extending the already existing ones. The degree of membrane model disruption correlated well with 1372


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Figure 9. Relative viabilities (in %) of human fibroblasts exposed to varying concentrations (10, 100, and 1000 μg/mL) of nanocapsules and biopolymer derivatives in solution for 24 h.

the levels of bacterial enzyme leakage and bactericidal effect against Escherichia coli. The large surface area to mass ratio coupled with high cationic charge of the NCs seemed to facilitate the interaction with bacterial membrane causing high local perturbance even in low NCs counts that did not induce cytotoxicity to human fibroblasts.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +34 93 739 85 70. Fax: +34 93 739 82 25. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.M.F. acknowledges the support of the European Commission under the Marie Curie Intra-European Fellowship (IEF) Program (Grant Agreement “NanoQuench” FP7-331416).

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