Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa

    Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa Parastoo Sabaeifard, Ahya Abdi-Ali, Mohammad Reza Soudi, Carlos Gamazo, Juan Manuel Irache PII: DOI: Reference:

S0928-0987(16)30337-2 doi: 10.1016/j.ejps.2016.08.049 PHASCI 3693

To appear in: Received date: Revised date: Accepted date:

1 June 2016 23 August 2016 25 August 2016

Please cite this article as: Sabaeifard, Parastoo, Abdi-Ali, Ahya, Soudi, Mohammad Reza, Gamazo, Carlos, Irache, Juan Manuel, Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa, (2016), doi: 10.1016/j.ejps.2016.08.049

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Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa Parastoo Sabaeifarda,b,c, Ahya Abdi-Alia, Mohammad Reza Soudia, Carlos Gamazob*, Juan Manuel

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Irachec a

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Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran. Department of Microbiology and Parasitology, University of Navarra , Pamplona, Spain.

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Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Pamplona, Spain.

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*Corresponding author: E-mail address: [email protected]; Tel.: + 34948425688, Ext. 806251; Fax: +34-948425649

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Abstract

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Amikacin is a very effective aminoglycoside antibiotic but according to its high toxicity, the use of this antibiotic has been limited. The aim of this study was to formulate and characterize amikacin loaded PLGA nanoparticles. Nanoparticles were synthetized using a solid-in-oil-in-water emulsion

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technique with different ratio of PLGA 50:50 (Resomer 502H) to drug (100:3.5, 80:3.5 and 60:3.5),

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two different concentrations of stabilizer (pluronic F68) (0.5% or 1%) and varied g forces to recover

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the final products. The most efficient formulation based on drug loading (26.0±1.3 µg/mg nanoparticle) and encapsulation efficiency (76.8±3.8%) was the one obtained with 100:3.5 PLGA:drug and 0.5% luronic F68, recovered by 20,000×g for 20 min. Drug release kinetic study indicated that

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about 50% of the encapsulated drug was released during the first hour of incubation in phospahte buffer, pH 7.4, 37 °C, 120 rpm. Using different cell viability/cytotoxicity assays, the optimized formulation showed no toxicity against RAW macrophages after 2 and 24 h of exposure. Furthermore, released drug was active and maintained its bactericidal activity against Pseudomonas aeruginosa in vitro. These results support the effective utilization of the PLGA nanoparticle formulation for amikacin in further in vivo studies. Keywords: Amikacin, PLGA, Optimization, Formulation, Pseudomonas aeruginosa

1. Introduction

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ACCEPTED MANUSCRIPT Pseudomonas aeruginosa is a Gram-negative bacterium which is extensively found in soil, water, plants and animals (Lang et al., 2004; Lyczak et al., 2000). It is one of the most important

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opportunistic pathogens and rarely causes disease in healthy persons (Abdi-Ali et al., 2006). However,

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the bacterium is easily able to infect immunocompromised and catheterized patients, patients with

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burn or traumatic wounds, malignancy, AIDS, cystic fibrosis (CF) and artificially ventilated individuals (Lang et al., 2004; Sabaeifard et al., 2014). Also, P. aeruginosa is considered as one of the main causes of nosocomial infections (Breidenstein et al., 2011a; Chakraborty et al., 2012). In

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addition, increasing the morbidity and mortality associated infections, this species is highly resistant to a variety of antibiotics (Breidenstein et al., 2011b; Chakraborty et al., 2012).

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Aminoglycosides is one of the most effective family of antibiotics generally used in the treatment of Gram-negative infections (Ghaffari et al., 2011; Jana and Deb, 2006). Particularly, amikacin is an

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anti-pseudomonas antibiotic and the second drug of choice in cystic fibrosis centers. Also, it is the

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preferred antibiotic in treatment of nosocomial infections (Jana and Deb, 2006). This drug binds irreversibly to 30S ribosomal subunit and inhibits an initiation complex formation with mRNA for

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protein synthesis, thus, prevent protein synthesis and results in cell death (Ehsan et al., 2014; Ghaffari et al., 2011; Jana and Deb, 2006; López-Díez et al., 2005). Besides, as being cationic, aminoglycoside

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antibiotics also cause membrane damages and altered ionic concentration (Jana and Deb, 2006; LópezDíez et al., 2005). However, due to their nephrotoxicity and ototoxicity, aminoglycosides are prescribed in limited and controlled doses (Abdollahi and Lotfipour, 2012; Ratjen et al., 2009; Zhang et al., 2010). According to their sub-micron size, nanoparticles (NPs) are able to efficiently cross biological barriers (Parveen et al., 2012). In addition, improved drug bioavailability and resistance time in the body, protecting the drug from degradation and gradual drug release pattern are other advantages of nanoparticles (Mudshinge et al., 2011; Parveen et al., 2012; Zhang et al., 2010). These traits result in decrease in drug amount, dose related toxicity and side effects and therefore make nanoparticles proper candidates to deliver toxic drugs (Mudshinge et al., 2011). Recently, an inhaled liposome delivery system has been reported to be used to reduce the drug toxicity while increasing drug efficacy 2

ACCEPTED MANUSCRIPT (Waters and Ratjen, 2014). Generally, the liposomal delivery systems are less stable in comparison to polymeric nanoparticles (Pinto-Alphandary et al., 2000). In this context, loading the drug in polymeric

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NPs could be of interest. To date, the only reported polymeric formulation loaded with amikacin has

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been synthetized by use of Eudragit®, while polymers like Poly (D, L-lactideco-glycolide) (PLGA) in

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spite of their unique characteristics, as biodegradability and biocompatibility, have not yet been used in the development of amikacin-loaded nanoparticles (Sharma et al., 2015). In the present study, we investigated whether the bactericidal effect of amikacin against P.

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aeruginosa biofilms could be increased by encapsulating the antibiotic in a new formulation based on PLGA nanoparticles. Therefore, we synthetized amikacin-loaded nanoparticles (A-NPs) with different

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ratios of PLGA 50:50 (502H) to drug and different concentrations of pluronic F68. Drug release kinetic studies and cytotoxicity assays were performed to select the optimal formulation to perform the

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cytotoxicity and bactericidal activity in vitro studies. The results obtained support the effective

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utilization of the PLGA nanoparticle formulation for the treatment of persistent Pseudomonas biofilm

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infections.

2. Materials and methods

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2.1. Materials

Amikacin hydrate was purchased by TOKU-E (Bellingham, USA). Poly (D, L-lactideco-glycolide) (PLGA) 50:50 (Resomer® RG 502H) was obtained from Boehringer-Ingelheim (Ingelheim, Germany). Fluoraldehyde™ O-phthalaldehyde Reagent Solution (OPA) was from Thermo scientific (Barcelona, Spain). 2-[N-morpholino] ethanesulfonic acid) monohydrate (MES) and Pluronic® F-68 were from Sigma-Aldrich Co. (St. Louis, USA). Acetone was purchased by VWR (Barcelona, Spain). Trypticase soy both (TSB) was purchased by BioMérieux (Marcy l’Etoile, France). Mueller Hinton broth II (cation adjusted) was from BBL™ (Maryland, USA).

2.2. Nanoparticles Preparation 2.2.1 Amikacin-loaded Nanoparticles (A-NPs)

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ACCEPTED MANUSCRIPT A-NPs were prepared by solid-in-oil-in-water method. Amikacin (3.5 mg) was dissolved in 100 µL ultrapure water. The drug solution was then added to 2 mL acetone containing varying amount of

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PLGA (60, 80 or 100 mg). The s/o phase was added to 10 mL pluronic F-68 (0.5% or 1% in 25 mM

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MES buffer) solution at pH 10. A-NPs were recovered by emulsions centrifugation at 20,000×g for 20

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min. Recovered A-NPs were freeze-dried in presence of 5% sucrose for 72 h.

2.2.2 Blank Nanoparticles (NPs)

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One hundred milligram PLGA was dissolved in 2 mL of acetone. One hundred microliters ultrapure water added to polymeric solution. Polymeric solution was then added to 10 mL pluronic F-

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68 (in 25 mM MES buffer) solution at pH 10, fully stirred at 300 rpm. NPs were recovered as

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described for A-NPs.

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2.3 Nanoparticles Characterization

2.3.1 Particle size and zeta potential

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Particle size and zeta potential of the formulations were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetaplus

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apparatus (Brookhaven Instrument Corporation, USA). The diameter of the particles was determined before and after lyophilization by dispersion of nanoparticles in ultrapure water. The measurements were done at 25 °C with a scattering angle of 90°. Zeta potential was measured after addition of 1 mM pH 6 KCl solution to above nanoparticles solutions (Penalva et al., 2015).

2.3.2 Drug Loading To determine the amount of drug encapsulated in 1 mg of A-NPs, the amount equal to 5 mg of each formulation was dissolved in 1 mL of 1M NaOH. The solutions were diluted 1:9 with 0.4 M boric acid pH 9.7. Fifty µL of the diluted samples was added to 50 µL of OPA reagent. Fluorescence was measured immediately at a λex/λem of 340/450 (Tecan GENios fluorimeter (Tecan Group Ltd, 4

ACCEPTED MANUSCRIPT Maennedorf, Switzerland)), respectively, and compared to a calibration curve of amikacin in 0.4 M boric acid pH 9.7 (Imbuluzqueta et al., 2011). Drug loading was calculated as the amount of drug (µg)

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per unit of mass of A-NPs (mg). The encapsulation efficiency was calculated as the ratio between the

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amount of entrapped drug and the initial amount of drug added and expressed in percentage.

2.3.3 Scanning Electron Microscopy (SEM)

A drop of freshly prepared A-NPs was placed onto a double-faced adhesive tape on a metal stub

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observed by SEM (Zeiss DSM 940A, Germany).

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and dried overnight. The samples were coated by gold to a 16 nm thickness (Emitek K550) and

2.3.4 Transmission Electron Microscopy (TEM)

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Two milligram of the sample was diluted with ultrapure water and 10 μl of the suspension was

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placed on a 200-mesh Formwar-coated copper grid (EMS, FF200-Cu). The grid was left for 30

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seconds at room temperature and washed 3 times with ultrapure water. Then, uranyl acetate (3%) was dropped onto the grids (5 min) to stain the sample. The images were captured using a 120 kV ZEISS

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Libra 120 electron microscope.

2.3.5 In vitro release profile Release profile from A-NPs was studied using a dialysis technique. The amount equal to 10 mg of the A-NPs was dispersed in 1 mL phosphate buffered saline (PBS) pH 7.4 and placed in a dialysis bag (Spectra/Por, molecular weight cutoff 12,000-14,000 Da). To keep the sink condition, dialysis bag was placed in 24 mL PBS. Incubating at 37 °C under orbital shaking, at pre-determined intervals (0.5, 1, 2, 3, 4, 6, 8, 10 and 22 h), the whole receiver compartment was collected and replaced with the equal amount of fresh PBS (Abdelghany et al., 2012).The amount of released drug was determined as described in section 2.3.2.

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ACCEPTED MANUSCRIPT 2.4 Cell Viability and Toxicity Assay 2.4.1 RAW 264.7 Cell Line Culture

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Cell viability assays were done using mouse macrophage cell line, RAW 264.7 (ATCC TIB-71).

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RAW macrophages were maintained in RPMI medium 1640, supplemented with 1% penicillin-

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streptomycin (10.000 U/mL, Gibco, CA, USA) and 10% fetal bovine serum (Gibco, CA, USA) at 37 °C in a humidified 5% CO2 atmosphere. Briefly, 105 cells perwell cells were seeded in 96-well plates and incubated for 24 h. Then, 100 µL of freshly prepared nanoparticles suspension and free drug

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solution in respective medium (0.01–1 mg/mL) were replaced with the old culture medium. Two sets of controls treated with culture medium (live control) and cells treated with Triton™ X-100 1% (w/v)

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in culture medium (dead control), respectively, were also included. After the incubation time, the culture medium was removed and the cells were washed twice with PBS. Cell viability assays were

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done after 2 and 24 h of exposure to formulations using the 3-[4,5-dimethylthiazol-2-yl]-3,5 diphenyl

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tetrazolium bromide (MTT) test (Mosmann, 1983), alamar blue (AB) test and the neutral red uptake (NRU) test (Borenfreund and Puerner, 1985). Cell toxicity was assayed by use of Lactate

controls.

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dehydrogenase (LDH). Cell viability and cytotoxicity were assessed as a percentage in relation to

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Each experiment was repeated six times from three independent incubation preparations.

2.4.2 MTT Cell Viability Assay MTT assay depends on the mitochondrial reductive capacity to metabolize the MTT salt to a colored formazan product. The assay was done by adding 100 μl of MTT (0.5 mg/mL in RPMI) to each well. The plate was incubated for 4 h at 37 °C, 5% CO2 in the dark. The MTT solution was discarded and formazan crystals were solubilized using 125 μl of dimethyl sulfoxide (Mosmann, 1983). The plate was shaken for 10 min at room temperature, and absorbance was measured at 540 nm using a microplate reader (Agilent, USA).

2.4.3 Alamar Blue (AB) Cell Viability Assay 6

ACCEPTED MANUSCRIPT AB is an important redox indicator which is used to assess metabolic activity and cellular health (Rampersad, 2012). To investigate the metabolic function of the cells after incubation, 100 μl of the

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AB reagent solution in RPMI (final concentration of 0.01 mg/mL) was added to each well and plates

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were incubated for 4 h at 37 °C, 5% CO2. Fluorescence was measured using a microplate fluorimeter

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(λex/λem of 540/580). .

2.4.4 Neutral Red Uptake (NRU) Assay

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Neutral red is a cationic dye that is taken up into the cells where it is then trapped inside the lysosomes. The stain is only taken up and kept by intact lysosomes present in healthy cells (Miller et

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al., 2015). 100 μl of neutral red solution (0.4% (w/v) diluted 1:80 in RPMI) was added to each well and the plates were incubated for 2 h at 37 °C, 5% CO2. Then, the wells were washed twice with PBS.

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The resulted precipitate was dissolved in 100 μl 50% ethanol containing 1% acetic acid. Absorbance

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was measured at 540 nm using a microplate reader (Borenfreund and Puerner, 1985).

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2.4.5 Lactate Dehydrogenase (LDH) Cytotoxicity Assay LDH is a stable cytosolic enzyme which is released due to membrane damage in cell membrane.

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The LDH cytotoxicity assay was performed following the instructions recommended for Roche Cytotoxicity Detection Kit PLUS (LDH) (Roche, Switzerland). Briefly, after incubation, the plate was centrifuged (524× g, 5 min) and 50 μl were collected and transferred to a new 96-well plate. 50 µL of the LDH cytotoxicity detection kit reaction mixture was added to each well, and absorbance (492 nm, 620 nm) was read after 10 min of incubation at room temperature in the dark. Cytotoxicity was determined as following:

Cytotoxicity (%) =

× 100

Values above 10% were considered as cytotoxicity.

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2.5 Antibiotic Susceptibility Testing

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2.5.1 Planktonic Cells

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To check the antimicrobial efficacy of the loaded drug, antibacterial susceptibility tests were done

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for A-NPs (4–512 μg/mL), free drug (4–512 μg/mL) and NPs with the same concentration of polymer and drug, respectively, too.

Antibacterial experiments were investigated using broth microdilution method (CLSI M07-A9)

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against P. aeruginosa PAO1 in 96-well microtiter plates. Briefly, known concentration of A-NPs dissolved in ultrapure water was added to Mueller Hinton Broth II (cation adjusted) containing P.

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aeruginosa PAO1. The inoculum concentration was 1 × 106 CFU/mL. Prepared microtiter plates were incubated at 37 °C for 24 h. MIC was determined as the lowest concentration that inhibits bacterial

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growth (Wikler, 2003).

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MBC was determined by cultivating 10 µL from wells without any visible growth of bacteria on TSA medium. TSA plates were incubated for 24 h at 37 °C. MBC was determined as the lowest

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concentration where no growth was observed. Considering the inoculum used, the MBC is defined as the lowest concentration that kills 99.9% of inoculum population.

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2.5.2 Biofilm cells

P. aeruginosa PAO1 biofilm susceptibility assay was done using the MBEC Assay for Physiology and Genetics (P&G) (Innovotech, Edmonton, Alberta, Canada). Diluted PA01 suspension (150 μL, 105 CFU/mL) was added to each well of a MBEC plate and incubated for 24 h at 37°C and 120 rpm to form biofilm on the pegs of the MBEC plate lid. After 24 h, pegs were immersed twice in sterile 0.9% NaCl (170 μL per well) to remove unattached bacteria. Then, they were challenged with 2-fold serial concentrations (4–512 μg/mL) of free amikacin or amikacin-loaded nanoparticles in 170 μL media (3 replicates per condition) and incubated as described. After treatment, pegs were rinsed as described and placed in a recovery plate containing 170 μL of sterile 0.9% NaCl per well. Biofilms were detached by ultrasonic treatment for 10 minutes. 10 µL of each well was cultured on a Tryptic Soy

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ACCEPTED MANUSCRIPT Agar (TSA) plate. The plates were incubated for 24 h at 37°C. The lowest concentration of treatment

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at which no growth was seen after 24 h was determined as the MBEC (Harrison et al., 2006).

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2.6 Data analysis

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All experiments were performed at least in triplicate in three independent experiments. Data were expressed as mean ± standard deviation (SD). Comparisons of two groups were made by ANOVA and

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Tukey’s test in Minitab 15 statistical software.

3. Results and discussion

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3.1 Effect of PLGA:Amikacin ratio and stabilizer concentration Amikacin is active against most Gram-negative bacteria and is effectively used for the treatment of

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cystic fibrosis and nosocomial infections. Despite the efficacy of the drug, its usage is limited because

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of ototoxicity and nephrotoxicity (Ghaffari et al., 2011; Peloquin et al., 2004). Drug delivery systems are able to increase the drug’s therapeutic index and also reduce its toxic effects. Literature describes

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several solid lipid nanoparticle and liposomal formulations for amikacin delivery (Atyabi et al., 2009; Clancy et al., 2013; Ghaffari et al., 2011; Losa et al., 1991). Liposomes are less stable in comparison

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with solid lipid nanoparticles and polymeric nanoparticles. On the other hand, solid lipid nanoparticles usually have low drug loading, and unpredictable drug release kinetic(Ghaffari et al., 2011). Polymeric nanoparticles constitute a good alternative to overcome these problems (Pinto-Alphandary et al., 2000; Zhang et al., 2010). Among polymers which are used to prepare polymeric nanoparticles, according to well-stablished biocompatibility and biodegradability, and also the FDA approval in many drug delivery systems, PLGA is the first choice (Cheow and Hadinoto, 2010). But still no amikacin-loaded PLGA formulations have been described. One of the methods which is used for encapsulating hydrophilic drugs in PLGA is the emulsion method (Vrignaud et al., 2011). There are several emulsion techniques to prepare PLGA nanoparticles (Sah and Sah, 2015). Due to simplicity and sonication-free process, in the present study the s/o/w method was successfully used but several parameters must be considered since they may affect the 9

ACCEPTED MANUSCRIPT characteristics of the final product. The polymer and stabilizer concentration are able to affect particle size distribution, therefore, different ratios of PLGA:drug (100:3.5, 80:3.5 and 60:3.5) and stabilizer

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concentrations (pluronic F68 0.5% or 1%) were used to encapsulate amikacin (Mora-Huertas et al.,

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2010). As shown in Figure 1, in the presence of 1% pluronic F68, the higher the amount of polymer,

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the bigger the particle size. Formulations prepared by 80:3.5 and 60:3.5 ratio of polymer:drug produced particles of 287.9±2.9 and 247.5±0.4 nm, respectively, significantly smaller than the ones which were synthetized by 100:3.5 ratio of PLGA:drug (368.8±4.4). This phenomenon can be due to

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the stress produced by fluids viscosity. Increasing polymer concentration can lead to increase in organic phase viscosity, reducing the net shear stress and formation of droplets with larger size and

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also it may lead to slower dispersion of drug-polymer solution into the aqueous phase and formation of bigger nanoparticles. This physicochemical effect could be also responsible of the concomitant

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increase in the polydispersity of samples with the increasing PLGA:drug ratio. Interestingly, a lower

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concentration of pluronic F68 leaded to bigger particles, probably due to the increase in surface tension, reducing the net shear stress during emulsification (Figure 1A). Increasing the polymer:drug

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ratio also caused a significant increase in drug loading and encapsulation efficiency, that also might be explained by the lower drug diffusion to the aqueous phase when the viscosity of organic phase

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increased .

However, in addition to the effect of pluronic (or other stabilizers such as PVA) on drug encapsulation, other factors may be also involved such as drug chemical nature and its polarity (MoraHuertas et al., 2010). In fact these emulsifying agents may form molecular micelles through interactions between hydrophobic parts of the stabilizer with the hydrophobic core of the NPs (Cooper and Harirforoosh, 2014). Italia et al. demonstrated that when the concentration of the stabilizer increases, entrapment of lipophilic drug increases (Italia et al., 2007). On the other hand, Cooper et al. showed that the high polarizability of a hydrophilic drug such as diclofenac (27.9 Å3) is able to effectively work against the micelle formation properties of stabilizer and results in a decrease in drug entrapment as stabilizer concentration increases (Cooper and Harirforoosh, 2014). Under our experimental conditions, the highest drug loading was detected with 100 and 80 mg of PLGA in 10

ACCEPTED MANUSCRIPT presence of 0.5% of pluronic F68 and the highest encapsulation efficiency (EE) was seen in 100:3.5 ratio of PLGA:drug in 0.5% pluronic F68. In other words, a rise in stabilizer concentration caused

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lower hydrophilic drug loading and EE (Figure 1C and 1D). In the present study, the very high

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molecular polarizability of amikacin (58.2 Å3) could explain the increased EE and drug loading in

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presence of lower concentration of pluronic F68.

Among the synthetized nanoparticles, due to a higher drug loading and EE and also less batch to batch variation, the selected formulation for the following studies was the one formulated with 100 mg

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of PLGA and 3.5 mg drug in presence of 0.5% pluronic F68.

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Table 1. Physicochemical characteristics of nanoparticles. Data expressed as mean ± SD (n = 3). Size

Z-potential

Drug Loading

EE

(mV)5

(µg/mg NP)

(%)6

-29.8±1.5

25.96±1.30

76.77±3.81

-42.9±1.5

-

-

PdI4

447±7

NPs2

340±5

0.24±0.02

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A-NPs1

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(nm)3

0.21±0.05

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A-NPs: Amikacin-loaded Nanoparticles, NPs: Blank Nanopartciles, PdI: Polydispersity Index, EE: Encapsulation Efficiency Particles’ size and Z-potential were determined by photon correlation spectroscopy and

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electrophoretic laser Doppler anemometry, respectively.

Table 1 shows the main physicochemical characteristics of the optimized nanoparticles. The entrapment of amikacin in PLGA nanoparticles significantly increased the mean size (340 vs 447 nm) and decreased the negative zeta potential (-43 vs -30 mV). This decrease on the negative surface charge of the resulting nanoparticles would be related to the hydrophilic character and positive charge of amikacin (Italia et al., 2007; Waters and Ratjen, 2014). Thus, during the formation of nanoparticles, amikacin would accumulate on areas close to the surface of the resulting nanocarriers and, as a consequence, the negative zeta potential of nanoparticles would be reduced. On the other hand, the amount of amikacin loaded was calculated to be close to 26 µg /mg with an encapsulation efficiency of 77%.

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ACCEPTED MANUSCRIPT The nanoparticles presented here displayed a similar capability to load amikacin compared to other nanocarriers described previously by other research groups and dedicated to the encapsulation of

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aminoglycosides. Thus, Abdelghany and co-workers reported a payload of about 22 µg/mg of

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gentamicin in PLGA nanoparticles. These nanoparticles were prepared with an emulsification method

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and displayed an improved antibiofilm activity in comparison with the free drug (Abdelghany et al., 2012). Imbuluzqueta et al. investigated the effect of gentamicin loaded PLGA nanoparticles against intracellular bacteria. They used the oil-in-water emulsion solvent evaporation method, but the

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potential (≈ -3.3 mV) (Imbuluzqueta et al., 2011).

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synthetized nanoparticles had high drug loading (≈ 23 µg/mg PLGA) but they still had very low zeta

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A

60:3.5

PLGA:Amikacin Ratio 80:3.5 100:3.5

0 Zeta Potential (mV)

B

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*

-10

Pluronic F68 (0.5%)

-20

Pluronic F68 (1%)

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*

-40 -50

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*

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25

*

20

*

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15

Pluronic F68 (0.5%)

10 0

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80:3.5 100:3.5 PLGA:Amikacin Ratio

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60:3.5

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Pluronic F68 (1%)

5

80

*

*

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60 40 20 0 60:3.5

Pluronic F68 (0.5%) Pluronic F68 (1%)

80:3.5 100:3.5 PLGA:Amikacin Ratio

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Encapsulation Efficiency (%)

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30

D

Drug Loading (ug/mg NP)

C

Figure 1. Influence of the stabilizer concentration and the polymer:drug ratio on the physicochemical

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characteristics of the resulting amikacin containing nanoparticles. A: mean particle size. B: zeta potential. C: amikacin loading. D: encapsulation efficiency. Data are expressed as average ± SD, n=3.

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*: P≤ 0.05, significantly different from other formulations.

3.2 Electron microscopy morphological study The morphological studies of the nanoparticles revealed that these carriers were spherical with smooth surface (Figure 2). By SEM, the apparent size of amikacin-loaded nanoparticles was found to be similar to the mean size calculated by photon correlation spectroscopy. The mean size obtained by TEM of the visualized nanoparticles was lower than from SEM or PCS. This fact may be probably be explained by a migration of the biggest particles during the preparation of the sample.

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ACCEPTED MANUSCRIPT B

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A

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loaded nanoparticles. Scale bars indicate 500 nm

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Figure 2. Scanning electron microscopy (A) and transmission electron microscopy (B) of amikacin-

3.3 In vitro release profile

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The amikacin release kinetic from the NPs showed a biphasic profile (Figure 3). Approximately

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40% of the drug was released within the first hour and the remaining in the next 9h. The high burst release in first hour could be due to the presence of drug on the surface or close to the surface of

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nanoparticles (Cheow and Hadinoto, 2010). As it was discussed above, according to nature of drug and polymer, it was expected to have a considerable amount of drug on the surface or close to surface

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of NPs which faces the aqueous phase, since the hydrophilic drug tries to expel from the hydrophobic matrix into aqueous phase during NPs synthesis (Cohen-Sela et al., 2009). The burst release is followed by a slower release which is caused by the amount of drug that is encapsulated inside the nanoparticles and released following either diffusion, bulk degradation or both (Mittal et al., 2007; Naraharisetti et al., 2005).

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100

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80

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60 40 20 0 0

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10

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Cumulative Drug Release (%)

120

15

20

25

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Time (h)

Figure 3. In vitro release studies of amikacin from nanoparticles. Data are expressed as mean +/-SD (n

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= 3).

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Despite of hydrophilicity of aminoglycosides, several studies showed a long sustained drug release

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(for at least a few days) from nanoparticles synthetized by PLGA 502H (Abdelghany et al., 2012; Cheow et al., 2010; Imbuluzqueta et al., 2011). Abdelghani et al. reported a sixteen days gentamicin

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release for PLGA NPs formulation prepared by s/o/w method (Abdelghany et al., 2012). Imbuluzqueta et al showed a ten weeks gentamicin release kinetic from PLGA nanoparticles (Imbuluzqueta et al.,

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2011). In contrast, the present formulation is able to release the total drug content in one day. A fast drug release from PLGA nanoparticle also have been reported by Cheow et al. for levofloxacin loaded nanoparticles prepared by double emulsion technique where about 40-50% of drug was released at 4h (Cheow and Hadinoto, 2010). The high burst release followed by a fast sustained release could be of favor for antimicrobial applications. Furthermore, the burst release could provide drug concentrations above the level which is needed to inhibit bacterial growth between dosings which could decrease the possibility of drug resistance occurrence (Cheow and Hadinoto, 2010).

3.4 Cell Viability and Toxicity Assays Three different assays were used to test the cell viability in RAW macrophages. The percent of cell viability was determined in comparison with non-treated cells. No significant differences were 15

ACCEPTED MANUSCRIPT observed as a function of A-NPs, NPs and free amikacin tested concentrations after 2 and 24 h of exposure to RAW macrophages (Figure 4).

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Using LDH assay no toxicity were determined for all the tested concentrations of A-NPs, NPs and

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120 100 80

A-NPs

60

NPs

40

Free amikacin

20 0

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NRU

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120 100

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A-NPs

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NPs

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Free amikacin

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1

0.01

0.05 0.1 0.5 Concentration (mg/ml)

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Alamar Blue

0.05 0.1 0.5 Concentration (mg/ml)

B

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0.01

Viability (% Control)

Viability (% Control)

A

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MTT

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free amikacin (Figure 5).

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Figure 4. Representative result of cell viability assays of amikacin-loaded nanoparticles (A-NPs, black bars), empty NPs (white bars) and free amikacin (gray bars) on mouse macrophages RAW 264.7 cells after 2 h (A) or 24 h (B). Macrophages were treated to different concentrations of the above treatments as indicated. Cell viability was determined by MTT reduction assay (MTT), alamar blue reduction assay (Alamar Blue), and neutral red uptake assay (NRU). *: P≤ 0.05, significantly different.

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Figure 5. Cytotoxicity assay of amikacin-loaded nanoparticles (A-NPs), NPs and free amikacin on mouse macrophages RAW 264.7 cells after 2h (A) and 24h (B). Macrophages were treated with

assay. *: P≤ 0.05, significantly different.

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3.5 Antibacterial activity of Nanoparticles

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different concentrations of A-NPs, NPs and free amikacin. Cell viability was determined by LDH

3.5.1 Planktonic cells

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Antibacterial activity of drug-loaded NPs, versus free amikacin, was assayed against P. aeruginosa

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PAO1 broth including blank NPs as a control. Blank NPs did not show any antibacterial activity. Drug-loaded NPs presented antibacterial activity, however, it was four times lower (MIC: 16 µg/mL

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and MBC: 32 µg/mL) than free drug (MIC: 4 µg/mL and MBC: 8 µg/mL) that could result from gradual drug release from NPs.

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The current antibiotic treatment alone is often inadequate to treat Pseudomonas infection. Besides, one major challenge in antibiotic based therapy is to reduce the risk of selecting resistant bacteria. An ideal drug delivery system should pose two important elements: controlled and targeted delivery. In this regard, A-NPs have the potential to be an effective drug delivery system. Although the in vitro studies indicate a lower activity than free drug, A-NP mediated amikacin delivery offers other advantages over the free drug, such as the controlled and sustained release of the drug at the site of infection, thus increasing the therapeutic efficiency of the drug, minimizing the systemic side effect and lowering the frequency of administration.

3.5.2 Biofilm cells

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ACCEPTED MANUSCRIPT P. aeruginosa (PA01) biofilms formed on polystyrene pegs of the Calgary Biofilm Device were used for all the biofilm studies. The biofilms were treated with either free amikacin or

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amikacin-loaded nanoparticles for 24 h. MBEC concentration was affected by amikacin

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encapsulation into the nanoparticles where the amikacin-loaded nanoparticles’ MBEC was

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determined as 512 μg/mL versus the free amikacin’s MBEC as 128 μg/mL. The much higher MBEC values (relative to MIC and MBC) determined for both treatments are likely due to

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biofilm structure and the interaction of the anionic matrix of biofilm with the cationic aminoglycoside, limiting the amount of free drug available to act against the bacteria hiding

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inside the biofilm (Khan et al., 2010). In any case, to assess the final effect of A-NP, further studies should be conducted in order to determine the effect on the viability of nonculturable

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bacteria by using confocal laser scanning microscopy after live/dead bacterial viability

4. Conclusion

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staining.

In the present study, we used a solid-in-oil-in-water emulsion method to formulate and optimize

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formulation of A-NPs. To this aim, distinct PLGA:drug ratio were tested in presence of different concentrations of stabilizer. The highest drug loading and EE was obtained with a 100:3.5 ratio of PLGA:drug in aqueous phase containing 0.5% pluronic F68. Drug release study showed that about 40% of the encapsulated drug is released in the first hour and the remained amount of drug will release in 24 h. Nanoparticles showed no cytotoxicity against mouse macrophages RAW 264.7. Antibacterial and antibiofilm studies showed that the drug released was active against P. aeruginosa in vitro. It can be concluded that the characteristics of optimized formulation make it a good candidate for antibacterial applications.

5. Acknowledgements

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ACCEPTED MANUSCRIPT The authors thank Dr. Edurne Imbuluzqueta (Department of Pharmacy and Pharmaceutical

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Technology, University of Navarra, Pamplona, Spain) for her precious guidance.

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PLGA:Amikacin Ratio 80:3.5 100:3.5

60:3.5

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*

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30 25 15 10 5 0

Encapsulation Efficiency (%)

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Pluronic F68 (1%)

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Drug Loading (ug/mg NP)

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Pluronic F68 (0.5%)

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Zeta Potential (mV)

0

60:3.5

Pluronic F68 (0.5%) Pluronic F68 (1%)

80:3.5 100:3.5 PLGA:Amikacin Ratio

80

* *

60 40

Pluronic F68 (0.5%)

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Pluronic F68 (1%)

0 60:3.5

80:3.5 100:3.5 PLGA:Amikacin Ratio

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ACCEPTED MANUSCRIPT Figure 1. Influence of the stabilizer concentration and the polymer:drug ratio on the physicochemical characteristics of the resulting amikacin containing nanoparticles. A: mean particle size. B: zeta

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*: P≤ 0.05, significantly different from other formulations.

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potential. C: amikacin loading. D: encapsulation efficiency. Data are expressed as average ± SD, n=3.

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Figure 2. Scanning electron microscopy (A) and transmission electron microscopy (B) of amikacin-

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loaded Nanoparticles. Scale bars indicate 500 nm

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100

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80 60 40 20

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Cumulative Drug Release (%)

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0 0

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Time (h)

Figure 3. In vitro release studies of amikacin from nanoparticles. Data are expressed as mean +/-SD (n

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100 80

A-NPs

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NPs

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Free amikacin

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60 40 20

0.05 0.1 0.5 Concentration (mg/ml)

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NRU

80

1

A-NPs NPs

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0.05 0.1 0.5 Concentration (mg/ml)

Free amikacin

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B

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100

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Alamar Blue

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B

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Viability (% Control)

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Viability (% Control)

MTT

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Figure 4. Representative result of cell viability assays of amikacin-loaded nanoparticles (A-NPs, black bars), empty NPs (white bars) and free amikacin (gray bars) on mouse macrophages RAW 264.7 cells after 2 h (A) or 24 h (B). Macrophages were treated to different concentrations of the above treatments as indicated. Cell viability was determined by MTT reduction assay (MTT), alamar blue reduction assay (Alamar Blue), and neutral red uptake assay (NRU). *: P≤ 0.05, significantly different.

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Figure 5. Cytotoxicity assay of amikacin-loaded nanoparticles (A-NPs), NPs and free amikacin on

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mouse macrophages RAW 264.7 cells after 2h (A) and 24h (B). Macrophages were treated with

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assay. *: P≤ 0.05, significantly different.

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different concentrations of A-NPs, NPs and free amikacin. Cell viability was determined by LDH

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Graphical abstract

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ACCEPTED MANUSCRIPT Tables:

(nm)3

PdI4

Z-potential

Drug Loading

(mV)5

(µg/mg NP)

(%)6

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Size

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Table 1. Physicochemical characteristics of nanoparticles. Data expressed as mean ± SD (n = 3). EE

447±7

0.24±0.02

-29.8±1.5

25.96±1.30

76.77±3.81

NPs2

340±5

0.21±0.05

-42.9±1.5

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-

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A-NPs1

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A-NPs: Amikacin-loaded Nanoparticles, NPs: Blank Nanopartciles, PdI: Polydispersity Index, EE: Encapsulation Efficiency Particles’ size and Z-potential were determined by photon correlation spectroscopy and electrophoretic laser Doppler anemometry, respectively.

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