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Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research paper

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Nanoparticle-mediated delivery of the antimicrobial peptide plectasin against Staphylococcus aureus in infected epithelial cells

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Jorrit Jeroen Water a, Simon Smart a,b, Henrik Franzyk c, Camilla Foged a, Hanne Mørck Nielsen a,⇑

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a

Section for Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Department of Pharmacy and Pharmacology, Faculty of Science, University of Bath, Bath, United Kingdom c Section for Natural Products Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark b

a r t i c l e

i n f o

Article history: Received 7 September 2014 Accepted in revised form 9 February 2015 Available online xxxx Keywords: Staphylococcus aureus Antimicrobial peptide Pulmonary drug delivery Polymeric nanoparticles PLGA Antibiotics Infected epithelial cells Calu-3 A549 THP-1

a b s t r a c t A number of pathogenic bacterial strains, such as Staphylococcus aureus, are difficult to kill with conventional antibiotics due to intracellular persistence in host airway epithelium. Designing drug delivery systems to deliver potent antimicrobial peptides (AMPs) intracellularly to the airway epithelial cells might thus be a promising approach to combat such infections. In this work, plectasin, which is a cationic AMP of the defensin class, was encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles using the double emulsion solvent evaporation method. The nanoparticles displayed high plectasin encapsulation efficiency (71–90%) and mediated release of the peptide over 24 h. The antimicrobial efficacy of the peptide-loaded nanoparticles was investigated using bronchiolar epithelial Calu-3 cell monolayers infected with S. aureus. The plectasin-loaded nanoparticles displayed improved efficacy as compared to non-encapsulated plectasin, while the eukaryotic cell viability was unaffected at the assayed concentrations. Further, the subcellular localization of the nanoparticles was assessed in different relevant cell lines. The nanoparticles were distributed in punctuate patterns intracellularly in Calu-3 epithelial cells and in THP-1 cells, whereas A549 cells did not show significant uptake of nanoparticles. Overall, encapsulation of plectasin into PLGA-based nanoparticles appears to be a viable strategy to improve the efficacy of plectasin against infections in epithelial tissues. Ó 2015 Published by Elsevier B.V.

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1. Introduction Bacterial infections constitute a global health threat and a burden to healthcare systems, primarily due to a rising incidence of nosocomial infections and the spread of multidrug-resistant bacteria [1]. This serious threat recently made the World Health Organization to express its concern for the reality of a post-antibiotic era in the 21st century [1]. Staphylococcus aureus (S. aureus) and its methicillin-resistant strains (MRSA) are the cause of a large fraction of infections in both hospital and community settings [2]. Moreover, an estimated 20% of the global population are persistent carriers of the pathogen while 60% are intermittent carriers [3]. S. aureus is the most common cause of soft tissue and skin infections [4] and other more severe infections such as endocarditis [5], osteomyelitis [6], bacteremia [7] and pneumonia [8]. Additionally, S. aureus is a prevalent pathogen in cystic fibrosis patients, in which it can colonize the lungs for prolonged peri⇑ Corresponding author. Section for Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark. Tel.: +45 35 33 63 46; fax: +45 35 33 60 30. E-mail address: [email protected] (H.M. Nielsen).

ods [9]. The persistence of S. aureus infections has in recent years been linked to the ability of the bacteria to invade host epithelial cells and survive antibiotic treatment in the intracellular environment [10] as also found for well-known intracellular bacteria such as Mycobacterium tuberculosis, Listeria monocytogenes and Salmonella spp. The exact mechanism of intracellular persistence of S. aureus is not fully understood and its elucidation is ongoing [10]. Notably, intracellular persistence of bacteria can decrease the therapeutic efficacy of antimicrobial compounds considerably due to poor uptake of many antibiotics into infected host cells. Furthermore, the increased enzymatic activity in the infected cells, higher clearance and/or altered antibacterial activity of antibiotics at the micro-environmental conditions (e.g. pH) inside the host cell may diminish their efficacy. Such reduced intracellular efficacy has been reported for many antibiotics commonly prescribed against S. aureus infections, e.g. azithromycin [11], vancomycin [12], oxacillin [13] and imipenem [13]. Thus, combined with the overall rapid rise in the occurrence of bacterial resistance, the need for novel treatment strategies is evident and this might be pursued by either developing new classes of antibiotics and/or improving drug delivery strategies.

http://dx.doi.org/10.1016/j.ejpb.2015.02.009 0939-6411/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: J.J. Water et al., Nanoparticle-mediated delivery of the antimicrobial peptide plectasin against Staphylococcus aureus in infected epithelial cells, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.02.009

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Antimicrobial peptides (AMPs) constitute a promising naturally derived new class of potential antibiotics as they are a part of the innate immune system of virtually all organisms, and they form the first line of defense against foreign pathogens including viruses, fungi, yeast, protozoa and bacteria [14]. Many AMPs are highly potent and associated with reduced potential for development of drug resistance [15] as compared to conventional small-molecule antibiotics currently applied in the clinic. Plectasin, a defensinclass peptide, has proved to be efficacious against several Grampositive bacterial strains [16,17]. Unlike many classical antibiotics [18] its activity toward intracellular S. aureus has been reported to be lower than against extracellular bacteria [19]. To overcome such reduced efficacy and improve the therapeutic potential of these next-generation antimicrobials, the present work explored the use of poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles to improve the cellular uptake of plectasin to treat S. aureus infections in epithelial cells. PLGA nanoparticles have been investigated for a variety of local drug delivery applications, i.e. topical [20], ocular [21] and pulmonary [22] delivery. Besides, nanoparticles have been explored for intracellular delivery of different therapeutic molecules including nucleic acids, proteins, peptides, small molecules and antimicrobial compounds such as gentamicin [23,24] and rifampicin [25]. Epithelia in the nasal cavity and lungs are both interesting targets for enhanced drug delivery. Nasal carriage is a known risk factor for secondary infections [26], and thus complete clearance of S. aureus in immunocompromised and post-surgical patients may be facilitated by a drug delivery system capable of eradicating intracellular bacteria. In the case of lung epithelia, pulmonary infections are the most common secondary nosocomial infection, and increased incidence of secondary infections has been linked to nasal carriage. This emphasizes the need for development of intracellularly active antibiotics. In the present work, we investigated the use of PLGA nanoparticles as a drug delivery system for plectasin with a focus on potential application in drug delivery to the epithelia in the airways. The physicochemical properties of plectasin-loaded PLGA nanoparticles, prepared by using the double emulsion method, were investigated with regard to uptake of the nanoparticles, subcellular localization in human epithelial cells and macrophages, and their effect was tested against S. aureus in infected airway epithelial cell monolayers.

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2. Materials and methods

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2.1. Materials

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Poly(L-lactide:glycolide molar ratio 75:25) (PLGA75) 20 kDa and polyvinyl alcohol (PVA) 403 were purchased from Kuraray Chemical Co. (Osaka, Japan). Plectasin GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY; 4.4 kDa was kindly provided by Novozymes A/S (Bagsværd, Denmark). Cell culturing materials and assay reagents, i.e. Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin (pen/strep), L-glutamine, fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), human serum albumin (HSA), phosphate-buffered saline (PBS), F-12K medium, RPMI1640 medium, Mueller–Hinton broth (MHI), gentamicin, Triton X-100, sodium dodecyl sulfate (SDS) and trypsin– EDTA were acquired from Sigma–Aldrich (St. Louis, MO, USA). Phorbol 12-myristate 13-acetate (PMA) and HEPES buffering salt were purchased from Fisher Scientific (Slangerup, Denmark) and AppliChem (Darmstadt, Germany), respectively. Solvents for analysis were of analytical grade and purchased from Merck (Darmstadt, Germany), ultra-pure water was generated using a Barnstead™ Nanopure™ machine (Thermo Fischer Scientific, Rockford, IL,

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USA). Low protein binding plastic ware was used whenever possible.

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2.2. RP-HPLC analysis of plectasin

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All concentration determinations of plectasin were performed using a Shimadzu RP-HPLC system (Shimadzu, Kyoto, Japan) with a C18 column (50 mm  4.6 mm, 2.6 lm, Kinetex, Phenomenex, Allerød, Denmark) at a constant flow of 1.85 mL/min measuring UV-absorbance at 214 nm. The mobile phase was composed of solvent A (5:95 (v/v) acetonitrile:water and 0.1% (v/v) TFA) and solvent B (95:5 (v/v) acetonitrile:water and 0.1% (v/v) TFA). All samples were run on a gradient from 10% to 40% B over 2 min at 35 °C. Calibration curves were established (n = 3) and the limits of detection (LOD) and quantification (LOQ) were determined to 0.4 lg/mL and 1.4 lg/mL, respectively.

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2.3. Nanoparticle preparation

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Plectasin-loaded nanoparticles were prepared at a theoretical drug loading of 0.625%, 1.25% and 2.5% (w/w, plectasin:PLGA75) using a water-in-oil-in-water (w/o/w) double emulsion–solvent evaporation method (n = 3). In short, 500 lL of an aqueous peptide stock solution or water (negative control) was added to 500 lL of a PLGA solution (60 mg/mL) in dichloromethane. The primary emulsion was formed by sonication for 60 s using a 600W UP100H ultrasonic processor (Hielscher Ultrasonics, Teltow, Germany) at 60% amplitude (samples kept on ice) after which 1 mL of 2% (w/v) PVA in water was added to the primary emulsion and whirl mixed for 1 min. Another round of sonication was performed to create the secondary emulsion (60 s at 60% amplitude), additional 5 mL of 2% (w/v) PVA in water was added, and the samples were stirred for 1 h at room temperature to complete solvent evaporation. Afterward, the nanoparticles were washed twice with water by subsequent centrifugation (22,000g, 12 min) and redispersed using whirl mixing and an ultrasonic water bath until complete dispersion was achieved. The nanoparticles were finally freezedried for 24 h after which they were stored at 20 °C until use. All experiments were conducted using the same batches of nanoparticles.

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2.4. Physicochemical characterization of nanoparticles

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2.4.1. Particle size and zeta potential Particle size and zeta potential measurements were performed before and after freeze-drying and subsequent redispersion by dynamic light scattering (DLS) (n = 3). Freeze-dried nanoparticles were dispersed in water (0.2 mg/mL) by whirl mixing while nonfreeze-dried particles were diluted 100-fold after final dispersion (0.3 mg/mL). The mean particle diameter (Z-average) and polydispersity index (PDI) were measured by intensity at k = 633 nm at 25 °C by using a Malvern NanoZS (Malvern Instruments, Worcestershire, UK) at a 173° scattering angle. Zeta potential measurements were performed using the Malvern NanoZS on the same batches of nanoparticle dispersions. The performance of the zeta cell and the instrument was verified before use, applying a zeta potential transfer standard purchased from the equipment manufacturer. Size measurements were verified regularly by using a polystyrene particle size standard. Data were analyzed using a standard t-test comparing plectasin-loaded against the non-loaded nanoparticles (P < 0.05).

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2.5. Drug loading and encapsulation efficiency

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The drug loading of plectasin in the PLGA nanoparticles was determined by measuring the amount of plectasin in the aqueous

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bulk phase after solvent evaporation and isolation of the nanoparticles. The difference between the stock and bulk plectasin concentration was used to calculate the drug loading in percent relative to the PLGA weight. No significant adsorption of plectasin to the test tubes was evident, and non-specific adsorption is thus not expected to influence data. Both the concentration of plectasin in the stock solution and the aqueous bulk phase were determined by RP-HPLC (n = 3). The encapsulation efficiency (EE%) was subsequently calculated according to Eq. (1):

Encapsulation efficiency ðEE%Þ 222

Measured drug loading ¼  100% Theoretical drug loading

ð1Þ

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2.6. Scanning electron microscopy

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The surface morphology of the nanoparticles was inspected using a scanning electron microscope (SEM) (FEI/Philips XL30 FEG, Hillsboro, OR, USA). The freeze-dried nanoparticles were either applied directly to double-sided carbon tape or drop-cast after redispersion in water and dried. Samples were subsequently mounted onto stubs, sputter-coated with a 4 nm layer of gold and imaged at an accelerating voltage of 3 kV. The images were recorded using the secondary electron detector and the Scandium software package (Olympus Soft Imaging Solutions, Münster, Germany).

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2.7. Release studies

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The release profiles of plectasin from the freeze-dried nanoparticle batches were obtained after redispersion of the nanoparticles (water bath sonication and whirl mixing) in acetate-buffered HBSS (25 mM, pH 5.0) to a final concentration of 10 mg/mL. The dispersions were incubated at 37 °C in a linear-shaking water bath (Grant Instruments, Cambridgeshire, UK) at 40 rpm. At predetermined sampling intervals, the dispersions were spun down by centrifugation at 22,000g for 12 min, and the supernatants were removed. After replacing the removed supernatants with fresh buffer, the nanoparticles were resuspended by whirl mixing and sonication in an ultrasonic water bath. The collected supernatants were stored at 20 °C until RP-HPLC analysis.

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2.8. Cell culture

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Calu-3 cells were maintained in DMEM supplemented with 2 mM L-glutamine, A549 cells were maintained in F-12K medium, and THP-1 cells were maintained in RPMI1640 medium supplemented with 2 mM L-glutamine. All culturing media did also contain pen/strep (10,000 U/mL penicillin, 100 lg/mL streptomycin) and 10% (v/v) FBS with the exception of the medium used for the bacteria-infected Calu-3 cells applied in the dose–response studies. Cells were grown in an atmosphere of humidified air with 5% CO2 at 37 °C. Calu-3 and A549 cells were detached from culturing flasks at 80–90% confluency by trypsin–EDTA treatment, while THP-1 cells were grown in suspension until a density of 1.0  106 cells/ mL. All cells were subcultured at a ratio of approximately 1:5 once a week.

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2.9. Cell viability

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Cell viability was assessed by using the MTS/PMS assay [27]. Calu-3 cells were seeded at a density of 80,000 cells/cm2 and cultured for 24 h in flat-bottomed 96-well MicroWell™ plates (NUNC, Roskilde, Denmark) to >80% confluency. Cells were washed twice with HEPES-buffered HBSS (10 mM) adjusted to pH 7.4.

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Nanoparticles dispersed in HEPES-buffered HBSS (10 mM, pH 7.4) were subsequently applied to the cell layers and incubated for 24 h. SDS (0.2% w/v) in water and cell culture medium was used as negative and positive controls, respectively. After 24 h, the dispersions were removed and a mixture of 240 lg/mL MTS (Promega, Madison, WI, USA) and 2.4 lg/mL of PMS (Sigma–Aldrich) in HBSS was applied to the cells that subsequently were incubated until an optical density of 0.8 for the positive control was reached. Optical density was measured at 492 nm by using a POLARstar Optima (GMB Labtech, Offenburg, Germany), and the viability was calculated according to Eq. (2):

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ðODtest:  ODpos:ctrl:Þ  100% Relative viability ð%Þ ¼ ðODneg:ctrl:  ODpos:ctrl:Þ

ð2Þ

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2.10. In vitro dose–response in infected Calu-3 monolayers

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Calu-3 cells were seeded 24 h prior to the experiment at a density of 1.9  105 cells per well in 24-well CorningÒ CostarÒ plates (Sigma–Aldrich). A culture of S. aureus (ATCC 25923, American Type Culture Collection, Manassas, VA, USA) grown overnight in MHI was washed and resuspended in DMEM with 10% (v/v) HSA, and then incubated for 45 min. After incubation, the bacterial culture was washed with PBS, and approximately 109 bacteria resuspended in DMEM were added to each well containing Calu-3 cells and incubated for 2 h to allow for internalization of the bacteria. Extracellular bacteria were subsequently killed by incubation with 50 lg/mL gentamicin for 45 min, after which dead cells were removed by washing with PBS. The samples in DMEM were added to the wells and incubated for 24 h. The following concentration ranges were tested; 0.1–8 lM plectasin and 0.025–4 lM encapsulated plectasin in nanoparticles. After incubation, the cells were lysed by using 0.1% (v/v) Triton X-100 in water, and serial dilutions were afterward plated on trypticase soy agar (Sigma–Aldrich) plates and the number of colony-forming units (CFUs) was determined after 24 h of incubation at 37 °C by manual counting. The CFU values were compared to CFU counts performed at t = 0 and normalized for the protein concentration per well as determined by using the Bicinchoninic acid assay (Thermo Fischer Scientific) according to the manufacturer´s specification. All experiments were conducted using triplicate samples over three consecutive passages of cells. Data were analyzed using GraphPad Prism 6.0 (San Diego, CA, USA) using the Hill equation to fit the experimental data. From the fitted results, the following parameters were calculated: Relative maximum efficacy (Emax), bacteriostatic concentration (Cstatic), the half maximal effective concentration (EC50) and the goodness of the fit (R2).

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2.11. Intracellular localization of nanoparticles

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Visualization of nanoparticle uptake in Calu-3, A549 and THP-1 cells was performed by using confocal scanning laser microscopy (CLSM) applying an inverted Leica SP2 confocal microscope (Wetzlar, Germany) equipped with a 63 (1.4 N.A.) water-immersion objective. Cells were seeded onto poly-D-lysine-coated 35 mm MatTek Glass Bottom Microwell Dishes (MatTek, Ashland, MA, USA) and grown until 80–90% confluency. THP-1 cells were cultured in the presence of 20 ng/mL of PMA to stimulate monocyte differentiation into macrophages and to create an adherent monolayer. The cells were rinsed with PBS and incubated for 1 h with 0.5 mg/mL PLGA nanoparticles in DMEM. The PLGA nanoparticles contained a theoretical drug loading of 0.625% plectasin and were fluorescently labeled by using 0.1% (w/w) 3,30 -dioctadecyloxacarbocyanine perchlorate (DiO) (Sigma–Aldrich) during particle

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preparation. After incubation, the cells were washed five times with PBS, and subsequently the following fluorescent probes were added according to the manufacturer´s protocols: 4.5 nM LysoTrackerÒ Red (Sigma–Aldrich), 3 lM DRAQ7™ nucleic acid live/dead stain (Biostatus, Leicestershire, UK), and 5 lg/mL Cellmask™ deep red (Life Technologies, Carlsbad, CA, USA) depending on the sample. After incubation, the cells were washed five times with ice-cold PBS and kept on ice until analysis.

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3. Results

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3.1. Plectasin encapsulation affects the size and zeta potential of the nanoparticles

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The nanoparticles obtained as dry powders by using the doubleemulsion solvent-evaporation method followed by freeze drying were spherical and within the nanometer range as observed from SEM images (Fig. 1a). The freeze-dried nanoparticle powders were fully redispersible independently of the drug loading as evident from the size measurements after redispersion (Table 1). The SEM data confirmed this (Fig. 1b), showing the presence of discrete nanoparticles in the redispersed samples. The freshly prepared and freeze-dried (and resuspended) samples had similar average hydrodynamic diameters around 220 nm, independently of the drug loading (Table 1). All samples had a mono-modal size distribution as indicated by the low PDI. The nanoparticle size was significantly decreased (P < 0.05, from 224 ± 3 nm to 215 ± 3 nm)

Fig. 1. Representative scanning electron micrographs of PLGA nanoparticles loaded with 0.625% plectasin. (A) Freeze-dried nanoparticle dry powder. (B) Nanoparticles redispersed in water. Scale bars equal 1 lm.

when the drug loading was increased (from 0% to 2.5%). The zeta potentials before and after freeze drying for plectasin-loaded nanoparticles increased significantly (P < 0.05) with increasing drug loading (Table 1), except for the nanoparticles with 0.6% drug loading as measured before freeze drying. At a 2.5% theoretical drug loading, the measured zeta potential was increased to less than half its initial negative value for the non-loaded particles. The encapsulation efficiency of plectasin in all nanoparticle batches was high, varying between 71.0 ± 0.3% and 90.6 ± 1.7%, and was inversely related to the theoretical loading (Table 1). The effective loading of plectasin in the nanoparticles reached a plateau with a maximum around 1.8% for 2.5% theoretical loading. Further increase of the theoretical drug loading led to a reduction of the encapsulation efficiency (data not shown). Optimizing the theoretical loading further was therefore not considered in this study.

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3.2. Drug loading-dependent release of plectasin

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The cumulative release of plectasin from the nanoparticles was assessed for 24 h (Fig. 2). In all cases, an initial burst release was observed, which appeared to be inversely related to the drug loading of the nanoparticles. At the lowest drug loading (0.6%), complete release of the peptide was observed already after 1 h. In case of nanoparticles with a 2.5% loading, the burst release was approximately 77 ± 3% of the encapsulated peptide followed by a trending continuous release of some of the remaining content during the following 24 h (up to 84 ± 6%). Release data were only gathered for 24 h, since this corresponds to the exposure time of the nanoparticles to bacteria in the in vitro dose–response assay.

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3.3. Nanoparticles do not reduce the viability of Calu-3 cells

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The impact of the PLGA nanoparticles on the viability of Calu-3 cells was determined by using the MTS/PMS assay. The nanoparticles were tested in equimolar plectasin concentrations as applied in the intracellular infection assay to investigate a possible influence of cellular toxicity on the results of the infection assay. These data show (Fig. 3) that both free plectasin and non-loaded PLGA nanoparticles did not exert any detectable cytotoxic effects on Calu-3 cells at the highest assayed concentration (4 lM). This is in line with reports in the literature where low toxicity was observed for PLGA nanoparticles loaded with different antimicrobial agents [28–30].

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3.4. Nanoparticles loaded with plectasin show improved dose– response against bacterial infection in vitro

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Dose–response relationships between plectasin and killing of S. aureus associated with Calu-3 monolayers were determined for a concentration range of free plectasin and equimolar concentrations of plectasin encapsulated and applied as PLGA nanoparticles. Analysis of the dose–response curves (Fig. 5) showed similar bactericidal activity of free plectasin, the control nanoparticles spiked with plectasin, and of 0.625% plectasin-loaded nanoparticles against S. aureus in the infected cell monolayers (Emax > 3 log). This observation is comparable with that of Lemaire et al. [13] showing similar bactericidal activity of (Emax  4) of the small molecule drug cloxacillin against S. aureus in infected Calu-3 cells. This killing effect (Emax) of plectasin expressed as 3.61 ± 0.39 [D log CFU (24h  0 h)] was not significantly affected after encapsulation of plectasin in PLGA nanoparticles and incubation with the infected cells for 24 h (Table 2). A trend of reduced Emax at increasing drug loading was observed for plectasin-loaded nanoparticles, but not for the spiked control samples, indicating that the uptake of plectasinloaded nanoparticles had a concomitant reduced Emax, that most

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J.J. Water et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx Table 1 Physico-chemical characterization of PLGA nanoparticles loaded with plectasin (mean ± SD, n = 3). Loading (%)

0.000 0.625 1.250 2.500 a b c d *

After freeze-dryinga

Before freeze-drying b

Size (nm)

PDI

223.8 ± 2.8 220.9 ± 4.5 218.3 ± 1.9* 215.8 ± 3.3*

0.09 ± 0.02 0.10 ± 0.02 0.07 ± 0.02 0.10 ± 0.03

f (mV)

c

39.1 ± 2.6 37.4 ± 2.1 33.0 ± 0.8* 22.8 ± 3.1*

Yield, encapsulation and effective loading b

Size (nm)

PDI

227.1 ± 3.0 221.7 ± 6.1 218.0 ± 3.9* 221.2 ± 3.1

0.09 ± 0.02 0.06 ± 0.01 0.08 ± 0.02 0.09 ± 0.03

c

f (mV)

Yield (%)

EE (%)d

Loading (%)

45.0 ± 2.2 38.4 ± 1.5* 31.5 ± 1.5* 18.0 ± 1.0*

78.7 ± 1.4 76.7 ± 0.7 79.0 ± 2.2 80.6 ± 1.5

n.a. 90.6 ± 1.7 85.1 ± 0.8 71.0 ± 0.3

n.a. 0.57 ± 0.01 1.06 ± 0.01 1.77 ± 0.01

After 90 days storage at 20 °C. Polydispersity index. Zeta potential. Encapsulation efficiency. Significant difference between PLGA-P 0% and sample determined by t-test (P < 0.05) only determined for size and zeta potential.

plectasin released (%)

110 100

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3.5. PLGA nanoparticles show endosomal escape

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To investigate further the intracellular fate of the nanoparticles, the uptake and intracellular localization of the nanoparticles was studied in vitro in different human airway epithelial cell lines, i.e. bronchial cells (Calu-3), alveolar cells (A549), as well as THP-1 macrophages by examining the co-localization between DiOlabeled plectasin-loaded nanoparticles (208.4 ± 5.5 nm) and LysoTrackerÒ (Fig. 4). After incubation for 1 h with the PLGA nanoparticles, uptake of high amounts was observed in both Calu-3 and THP-1 cells for both plectasin-loaded and non-loaded PLGA nanoparticles. In contrast, A549 cells showed only minor signs of uptake after incubation for 1 h, and no apparent difference was seen between loaded and non-loaded nanoparticles. THP-1 cells showed uptake of the PLGA nanoparticles. However, there was no observed difference in the uptake of loaded and non-loaded nanoparticles. THP-1 cells have been shown to internalize negatively charged polystyrene particles via clathrin-dependent endocytosis [31]. No co-localization of the PLGA nanoparticles with LysoTrackerÒ was apparent, neither in THP-1 nor in Calu-3 cells. This indicates that the particles did not reside in late endosomes or lysosomes inside the cells at the investigated time points.

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4. Discussion

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To enhance the amount of plectasin delivered with PLGA nanoparticles to the airways, the drug loading was initially optimized. The highest drug loading obtainable by using the investigated formulation parameters was 1.8% for a theoretical drug loading of 2.5%. Further increase of the theoretical loading resulted in an even lower actual drug load under the given conditions, and 1.8% was accepted as the maximum achievable drug load. This could be caused by saturation of the oil–water interface with peptide followed by limited localization in the nanoparticle core with predominant localization on the surface of the nanoparticles, which is also supported by the fact that the zeta potential of the plectasin-loaded nanoparticles was significantly increased (P < 0.05) at higher drug loading. Similar findings on predominant localization of protein on a hydrophobic nanoparticle surface have been reported [32]. Combined with the positive charge density of plectasin (between +1 and +3, depending on the pH [17]), this results in an increased nanoparticle zeta potential, as described previously for polymeric nanoparticles with the cationic additive dioleoyltrimethylammoniumpropane (DOTAP) [33,34]. The release profile of plectasin showed a significant burst release as is often observed from hydrophobic polymeric

450

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90 80 70 0

10

20

30

time (h) Fig. 2. Cumulative release of plectasin (normalized to drug loading) from PLGA nanoparticles dispersed in acetate-buffered HBSS (25 mM, pH 5.0). PLGA nanoparticles loaded with 0.625% (triangles), 1.25% (squares) and 2.5% (circles) plectasin (means ± SD, n = 3).

140 120

cell viability (%)

nanoparticles. Control experiments supported this finding, as the effect of spiking PLGA nanoparticle dispersions with free plectasin did not significantly reduce the EC50 or the Cstatic values.

100 80 60 40

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20 % -P A

-P A

PL G

1.

25

2. 5

%

% 0. 6 G

-P

-P PL

A PL G

PL G A

pl ec t

as

in

0%

0

Fig. 3. Cell viability of Calu-3 cells incubated with nanoparticle dispersions for 24 h. The amount of nanoparticles is adjusted to a total of 4 lM of plectasin per well. The applied nanoparticles weight for the non-loaded PLGA nanoparticles correspond to the amount of PLGA nanoparticles loaded with 0.625% plectasin (means ± SD, n = 3).

412 413 414 415 416 417 418 419 420 421 422 423

likely is caused by a prolonged release of plectasin from the nanoparticles with the high drug loading. The dose–response profile was improved by encapsulation of plectasin as both the EC50 and the Cstatic concentrations were more favorable as compared to those of the PLGA nanoparticle samples spiked with plectasin (Table 2). The EC50 value was reduced significantly from 1.24 ± 0.15 lM for free plectasin to 0.80 ± 0.12 lM for the nanoparticles loaded with 2.5% plectasin (P < 0.05), showing an improved efficacy. This indicates that encapsulation promotes the delivery of plectasin to bacteria thereby increasing bacterial killing. A similar reduction of the bacteriostatic concentration (Cstatic) was observed upon encapsulation of the AMP in

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Fig. 4. Confocal images of different cell lines incubated with PLGA nanoparticles; non-loaded (control) and loaded with 0.625% plectasin for 1 h. Images are z-slices selected from recorded z-stacks, representing the middle of the cell layer. Each row represents the same sample and the columns represent the different fluorescent channels (shown in gray scale). The right column shows an overlay of PLGA nanoparticles (green) and lysosomes (red). Scale bars equal 10 lm. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Δ log CFU (24h-0h)

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(A)

2

0

-2

0.2

0.4

0.8 1

2

3

4

plectasin concentration (μM)

Δ log CFU (24h-0h)

4

(B)

2

0

-2

0.2

0.4

0.8 1

2

3

4

plectasin concentration (μM) Fig. 5. Dose–response curves (adjusted for total release of plectasin after 24 h) of plectasin-loaded PLGA nanoparticles against S. aureus infected Calu-3 monolayers. For better visualization, four data points outside the axis limits are not shown (means ± SD, n = 3). (A) PLGA nanoparticles loaded with 0.625% plectasin (squares), non-loaded PLGA nanoparticle + 0.625% plectasin (closed triangles), plectasin (open squares) and non-loaded PLGA nanoparticles at equivalent weight dose of PLGA nanoparticles loaded with 0.6% plectasin (open triangles). (B) PLGA nanoparticles loaded with 2.5% plectasin (squares), non-loaded PLGA nanoparticle + 2.5% plectasin (closed triangles), plectasin (open squares) and non-loaded PLGA nanoparticles at equivalent weight dose of PLGA nanoparticles loaded with 2.5% plectasin (open triangles).

Table 2 Regression analysis of fitted dose–response curves. Data are presented as means ± SD, n = 3. Loading (%)

Emax (D log CFU (24h  0 h))

EC50 (lM)

Cstatic (lM)

R2

Plectasin 0.625% 2.5% 0% + 0.6% plectasina 0% + 2.5% plectasina

3.61 ± 0.39 3.37 ± 0.62 2.90 ± 0.47* 3.75 ± 0.46

1.24 ± 0.15 0.96 ± 0.14 0.80 ± 0.12* 1.22 ± 0.28

1.48 1.21 1.08 1.51

0.92 0.78 0.81 0.94

3.63 ± 0.45

1.21 ± 0.43

1.43

0.91

a

Samples consisted of non-loaded PLGA nanoparticles according to the loaded sample weight, spiked with plectasin. * Significant difference between non-loaded PLGA nanoparticles and sample determined by t-test (P < 0.05) only determined for size and zeta potential.

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nanoparticulate drug delivery systems encapsulating hydrophilic compounds [35]. The release from the PLGA nanoparticles was dependent on the drug loading, leading to a prolonged release at the highest theoretical drug loading (2.5%). This could be explained by a shift in the ratio between encapsulated and emulsion interface-associated plectasin at higher drug loading. In case of a high affinity of plectasin for the emulsion interface, a low drug loading would mainly result in surface-associated plectasin [36] with a higher burst release, whereas a higher drug loading will lead to saturation of the interface and consequently a higher encapsulation in the core of the nanoparticles. This encapsulated plectasin

7

will be released more slowly as shown for the prolonged release of plectasin from nanoparticles loaded with 2.5% drug. The burst effect may have had a possible influence on the efficacy measured in the in vitro infection cell assay, as the burst effect occurs instantaneously upon redispersion of the nanoparticle powder. However, a significant and relatively fast uptake of the nanoparticles would occur on a minute to hour timescale, as previously shown for uptake of anionic polymeric nanoparticles into eukaryotic cells, presumably by clathrin-mediated endocytosis [37], which occurs at a higher uptake rate than adsorptive endocytosis and caveolin-dependent endocytosis [37]. Consequently, nanoparticles with a high burst release corresponding to almost 100% of the loaded peptide would not give rise to a sustained release of plectasin after cellular uptake, leading to lower intracellular accumulation as found for other drug-loaded polymeric microparticles tested in macrophages [38,39]. This alters the ratio between the extracellular and intracellular plectasin concentrations in the assay and would thus influence the killing effects on the bacteria residing intracellularly. The effect was observed in the in vitro assay, as the relative improvement of the efficacy (EC50) of the nanoparticles appeared to be plectasin-load dependent, with increased drug loading leading to a larger reduction of the EC50 concentration when the same total plectasin dose was applied to the cells. Increased intracellular accumulation of plectasin due to a lower burst release together with a more prolonged release thus might improve the killing of intracellular S. aureus inside Calu-3 cells. As mentioned above, plectasin-loaded nanoparticles mediated a reduction of the EC50 as compared to non-encapsulated plectasin in the S. aureus infected Calu-3 monolayers. A similar improvement was found for the Cstatic. In contrast, the Emax values were reduced for plectasin-loaded formulations, indicating a lower overall reduction of the CFU counts over 24 h. As the reduction appeared to be dependent on the drug loading this reduction could be influenced by the different release profiles of the plectasin-loaded nanoparticles, with prolonged release having an overall decreased killing effect on S. aureus over 24 h. To ensure that the antimicrobial effect was mediated by nanoparticle uptake into the Calu-3 monolayer, this was evaluated by using confocal microscopy. In addition to the Calu-3 human airway epithelial cells representative of bronchial cells present in the airways, another cell line, namely, A549 cells were investigated as alveolar type epithelial cells present in the airways. Finally, the uptake in THP-1 macrophages was investigated, as this is the predominant immune cell present as a first line of defense against pathogens in the lungs. The Calu-3 cells seemed to have a higher uptake of the plectasin-loaded nanoparticles as compared to nonloaded nanoparticles. This could be attributed to non-specific interactions between nanoparticle surface-associated plectasin and the Calu-3 cell membrane or membrane components. Plectasin has been shown to bind specifically to the second sugar in Lipid II (i.e. the N-acetyl glucosamine) [40]. Lipid II is a bacterial cell wall precursor in Gram-positive bacteria while N-acetyl glucosamine is a common monosaccharide present on eukaryotic cell membranes as well. Binding to N-acetyl glucosamine leading to increased uptake in Calu-3 cells has been shown for wheat germ agglutinin-conjugated PLGA nanoparticles [41]. The THP-1 cells tended to take up both plectasin-loaded and non-loaded nanoparticles. For the A549 cells, a reduced uptake was observed as compared to that of the Calu-3 cells, which is likely caused by the fact that the uptake of PLGA nanoparticles is mainly driven by adsorptive endocytosis, as described by Tahara et al. [42], and which proceeds with a lower uptake rate as compared to clathrin-mediated endocytosis [37]. Consequently, a lower uptake in A549 cells was observed after 1 h as compared to Calu-3 and THP-1 cells, which are more prone to clathrin-mediated uptake

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[31,41]. However, further studies are needed to clarify the uptake mechanisms. In both Calu-3 and A549 cells, no co-localization with LysoTrackerÒ was observed, indicating that the nanoparticles either reside in the early endosomes or escape from the endo-lysosomal pathway, independently of the presence of plectasin. This is in line with several reports in the literature observing endosomal escape of PLGA microparticles in macrophages [43], as well as of nanoparticles in epithelial cells [44] and human smooth muscle cells [45,46]. Endosomal escape has been reported to occur within 10 min after uptake [47], unlike other nanoparticles such as quantum dots, silica, gold and chitosan-based nanoparticles that are retained in the lysosomes after endocytosis [48] resulting in degradation of the AMPs. Such endosomal escape would be beneficial due to the direct co-localization of plectasin with the intracellularly residing bacteria, since S. aureus has been shown to reside in the cytoplasm in case of human epithelial 293 cells [49] and tracheal epithelial cells [50,51]. However, the subcellular localization has been shown to be highly dependent on the bacterial strain and the host cells, with reported localization ranging from cytoplasmic, vacuolar, endosomal, phagolysosomal and to the autophagic pathway [52,10]. Finally, endosomal escape could potentially promote cellular retention of plectasin due to improved stability after escape from the lysosomal degradation pathway. Further studies are required to investigate the in vivo effect of plectasin-loaded nanoparticles on S. aureus infections.

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5. Conclusion

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The present work demonstrates the potential of plectasin-loaded PLGA nanoparticles that may be taken up by human lung epithelial cells as well as by macrophages in relation to treatment of airway S. aureus infections. Distinct cellular uptake patterns of PLGA nanoparticles were observed for the different epithelial cell lines, highlighting the possible delivery challenges in in vivo settings, as efficacy will be highly dependent on the subcellular S. aureus localization. However, the PLGA nanoparticles showed a significant increase in efficacy (EC50) of plectasin in the in vitro infection cell model. This shows the potential of PLGA nanoparticles as a drug delivery system to increase the efficacy of antibiotics and AMPs against bacteria residing intracellularly and extracellularly.

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Acknowledgments

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The authors acknowledge Thara Qais Hussein and Maria Læssøe Pedersen for maintaining the cell lines used in this study. The Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen is acknowledged for use of their imaging facilities and The Danish National Advanced Technology Foundation is acknowledged for co-financing the HPLC system. Novozymes A/S is acknowledged for supplying the antimicrobial peptide and the Danish Agency for Science and Technology and Innovation (DanCARD, grant no. 06-097075) for financial support.

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