Erythromycin encapsulation in nanoemulsion-based delivery systems for treatment of Helicobacter pylori infection: Protection and synergy

Accepted Manuscript Erythromycin encapsulation in nanoemulsion-based delivery systems for treatment of Helicobacter pylori infection: Protection and synergy Le Tuyet Chau Tran, Claire Gueutin, Ghislaine Frebourg, Christophe Burucoa, Vincent Faivre PII:

S0006-291X(17)31820-X

DOI:

10.1016/j.bbrc.2017.09.060

Reference:

YBBRC 38505

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 24 August 2017 Accepted Date: 12 September 2017

Please cite this article as: L.T.C. Tran, C. Gueutin, G. Frebourg, C. Burucoa, V. Faivre, Erythromycin encapsulation in nanoemulsion-based delivery systems for treatment of Helicobacter pylori infection: Protection and synergy, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.09.060. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Erythromycin encapsulation in nanoemulsion-based delivery systems for treatment of Helicobacter pylori infection : protection and synergy. Le Tuyet Chau TRAN1, Claire GUEUTIN1, Ghislaine FREBOURG2, Christophe BURUCOA3, Vincent FAIVRE1*

Institut Galien Paris-Sud, CNRS, Université Paris-Saclay, Univ. Paris-Sud; 5, rue Jean-Baptiste Clément, Châtenay-Malabry 92296, France.

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1

Institut de Biologie Paris-Seine (IBPS), Université Pierre et Marie CURIE; SME-9 Quai St Bernard, 75252 Paris Cedex 05, France 3

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EA 4331 LITEC, Université de Poitiers, CHU de Poitiers; 40, avenue du Recteur Pineau, 86022 Poitiers Cedex, France

Abstract

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Poorly water-soluble and unstable compounds are difficult to develop as drug products using conventional formulation techniques. The aim of the present study was to develop and evaluate a nanoformulation prepared by a hot high-pressure homogenization method, which was a scalable and solvent-free process. We successfully prepared stable nanodispersions to protect a labile antibiotic, erythromycin. The mean diameter of the dispersed droplets was

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approximately 150 nm, and size distribution was unimodal. Dispersion was physically stable at room temperature for over six months. Using erythromycin as a model compound, we studied its antimicrobial activity in vitro on Helicobacter pylori. Results showed that drug encapsulation improves API stability in an acidic environment and is conducive to

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synergistic effect between the drug and the formulation.

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Keywords: Erythromycin, Helicobacter pylori, nanoemulsion, high pressure homogenization.

* Corresponding author Tel.: +33 146835465; Fax: +33 146835312 E-mail address: [email protected] (Vincent FAIVRE)

ACCEPTED MANUSCRIPT Introduction

1.

Since 1982, when Helicobacter pylori (H. pylori) was first discovered by Warren and Marshall[1], scientists amassed much information about this Gram-negative microaerophilic bacterium, which is commonly found in the stomach of approximately one half of the world's population, and its associated pathologies[2]. H. pylori is the cause of most peptic ulcer disease

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and a primary risk factor for gastric cancer[3]. Numerous recommendations have been proposed for the treatment of H. pylori infection with combinations of antimicrobials as the first choice for patients carrying the infection with peptic ulcer disease and gastric MALTlymphoma[4-6].

Macrolides

are

interesting

therapeutic

agents

used

in

treating H.

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pylori infections. Among them, clarithromycin is more acid-stable and consequently clinically most effective[7]. Standard triple therapy composed of proton pump inhibitor (PPI), clarithromycin and amoxicillin/or metronidazole is more successful if extended to fourteen

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days. Increased resistance to the antibiotics used in PPI triple therapy needs to be considered in the selection of treatment. The resistance of H. pylori strains to clarithromycin and/or metronidazole is an important problem and its clinical relevance has been conclusively shown [8, 9]

.

It is consequently important to develop new entities against H. pylori and thereby enlarge the

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treatment panel. However, the main limitation of this development consists in the chemical properties of the gastric medium, particularly its strong acidity. Indeed, H. pylori are sensitive to many antibiotics but due to chemical degradation most of them cannot be used in an acidic medium[10]. When it is prescribed, clarithromycin degradation half-life has been calculated at

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around 15.8 and 96.7 h at pH 3 and 4 respectively, but at pH 2, it decreases to 1.3 h[11, 12]. Its activity results mainly from re-excretion of the absorbed fraction, which is e why a large

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amount must be administered. In patients with gastro-duodenal ulcers due to H. pylori infection, clarithromycin is given twice daily in a dosage of 500 mg [13]. Regarding the survival mechanism of bacteria in the stomach, a local delivery of active pharmaceutical ingredients (API) close to the bacteria could be advantageous. Due to the activity of urease, the environment of the bacterium is neutralized by the production of ammonia and carbon dioxide[14]. Hence, a release of the active substance near the bacterium should overcome the problem of acidity. Encapsulation of an active substance could be a good approach by offering protection against stomach acidity. Nanoemulsions have been selected as they are easy to produce, capable of protecting labile APIs, and small enough to diffuse into mucus; moreover, some lipids have antibacterial activities[15].

ACCEPTED MANUSCRIPT Erythromycin, the first macrolide antibiotic, which has an effect on H. pylori in vitro but is not used in treatment because of its strong acid-lability, has been selected as a model drug. The objective of this study was to design and evaluate a delivery system for a potential oral formulation of erythromycin. Firstly, erythromycin-loaded nanodispersions (ERY-loaded Nds) were developed and characterized. Secondly, the formulation was evaluated as concerns its in

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vitro antimicrobial properties. Materials and methods

2.1. Reagents and chemicals

glycerides)

were

kindly

provided

by

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Gelucire®50/13 (stearoyl macrogolglycerides), Labrafil®M2125CS (linoleoyl polyoxylGattefosse

S.A.S.

(Saint-Priest,

France).

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Phospholipon®90G (PL90G: soybean lecithin at 94–102% of phosphotidylcholine) was offered from Lipoid Group (Köln, Germany). Erythromycin were purchased from SigmaAldrich (France). HPLC grade acetonitrile and methanol, together with analytical grade potassium dihydrogen phosphate, were purchased from Carlo Erba (Italy). The water used for all experiments was purified water obtained from a Milli-Q system (Millipore, France). All other chemicals were of analytical grade or reagent grade.

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2.2. Preformulation studies

2.2.1. Development the HPLC analytical method for erythromycin and Labrafil®M2125CS High-performance liquid chromatography (Waters Corporation, USA) equipped with a Water

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600 Controller-Millipore pump, a Water 717 plus Automated sample injector, and a Water 2996 Photodiode Array Detector set at 215 nm were used. A reversed-phase column (YMC-

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Triart C18, 3 µm, 150 x 4.6 mm I.D.) with a 10 x 4.0 mm I.D. stainless-steel pre-column was also used. The temperature of the column was maintained at 50 °C. The mobile phase, consisting of acetonitrile-phosphate buffer pH 9.0-water (45:6:49), was filtered through a 0.22 µm GS-type filter (Millipore, Ireland), degassed under vacuum prior to use and used at flow rate of 1.0 ml/min. The working standard solution of erythromycin A (10 mg/g) was prepared in a volumetric flask by dissolving erythromycin A in a small portion of acetonitrile, and then diluting with the mobile phase. The quantification of erythromycin A was carried out in a range from 0.2 to 15.0 mg/g (r2=0.9992; Accuracy: recovery >99%; Repeatability: %RSD = 0.61). Concerning erythromycin-loaded nanodispersions (ERY- NDs) a portion was weighed and dissolved in acetonitrile-methanol (50:50) at concentration in the linearity plot. The

ACCEPTED MANUSCRIPT mixture was sonicated for 10 min, followed by ultracentrifugation 30,000 rpm at 4 °C for 1 hour. The supernatant was then filtered through a 0.20 µm RC-membrane (Minisart, Germany).Quantification of Labrafil®M2125CS was carried out in a range from 0.5 to 10 mg/g with (r2=0.9992) by the HPLC procedure mentioned above. The retention time of maximum peak corresponding to Labrafil®M2125CS was ~13 min.

corresponded to the maximum peak in Labrafil®M2125CS, the

procedure could be assayed in the formulation for this lipid. 2.2.2. The solubility of erythromycin in water

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determine exactly what

While we did not

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The solubility of erythromycin in water was determined by adding an excess of the drug into water. The suspensions were stirred by a magnetic stirrer at 25 °C for 24 h. The supernatant was filtered through a Minisart (RC25, 0.20 µm, Sartorius, Germany). The filtrate was immediately

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diluted by mobile phase, and the content of dissolved erythromycin was analyzed by high pressure liquid chromatography (HPLC). All experiments were conducted in triplicate. 2.2.3. The solubility of erythromycin in lipid excipients

The solubility of API in lipid-based excipients was studied to determine the most suitable lipid for

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incorporation of API into the carrier. This was performed by dissolving increasing amounts of API in various melted solid or liquid lipids, and determining the maximum percentage of API that could be dissolved in each lipid through the naked eye. The solubility of erythromycin in the most suitable lipid was then tested by HPLC.

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A given amount of erythromycin was dissolved in lipid and stirred for 4 hours at 65-70 °C, then ultra-centrifuged at 30,000 rpm for 60 min. The supernatant was diluted by acetonitrile,

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ultra-centrifuged at 20,000 rpm for 30 min, was diluted by mobile phase, and filtered through a 0.20 µm RC-membrane (Minisart, Germany). The filtrate was immediately assayed by HPLC. All the data are the mean of three separate experiments. 2.2.4. The stability of erythromycin in Labrafil®M2125CS The study was carried out by dissolving 5 mg of erythromycin in 1 g of Labrafil®M2125CS at 37 °C under constant magnetic stirrer (800 rpm) in a heated bath. Drawn samples at 1, 3, 6, 9, 12 and 24 h were diluted in suitable amount of acetonitrile, and put in ultra-sound for 2 min. The sample was then diluted in mobile phase and filtered through a 0.20 µm RC-membrane (Minisart, Germany). The content of erythromycin was analyzed by HPLC in triplicate.

ACCEPTED MANUSCRIPT 2.3. Preparation of erythromycin loaded nanodispersions The lipid nanodispersions were produced by high pressure homogenization. Briefly, erythromycin was dissolved in the molten lipid phase for 4 hours at 65-70 °C. A pre-heated aqueous phase containing 2% (w/w) of Gelucire®50/13 and 1% (w/w) of PL90G as surfactant

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was formed by using ultra-turrax (IKA®T18, Germany) at 11,000 rpm for 5 min. Thereafter, the lipid phase (20% w/w) containing or not the drug (concentrations from 1 to 10wt% of lipid phase) was added to the water phase and formed a pre-emulsion at 20,000 rpm for 5 min. The resulting dispersion was homogenized using a two stage high-pressure homogenizer (APV-2000, Denmark) during 5 min at 70°C. The pressure was constantly kept at 600 and

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200 bars, in the first and second stages respectively during the whole process. The

2.4. Particle size measurements

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formulation was stored in glass vials and cooled down slowly to room temperature (RT).

Particle sizes were determined by dynamic light scattering using a Zetasizer Nano-ZS90 (Malvern Instruments, UK). Particle size was evaluated by the z-average diameter and size distribution was described by the polydispersity index (PdI). The dispersions were three

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hundred-fold diluted in Milli-Q water. Measurements were taken on three different batches. Cryo – Transmission Electron Microscopy (Cryo-TEM)

2.5.

The morphology of ERY-loaded NDs was determined by cryo-TEM. The sample was

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prepared by placing a drop of this system, which was diluted 40-fold (v/v) in Milli-Q water onto a copper grid and blotted with filter paper to form a thin liquid film. The thinned sample

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was plunged into liquid ethane at its freezing temperature (-183 °C) to form a vitrified specimen and subsequently transferred to liquid nitrogen (-196 °C) for storage. The vitrified specimens were examined with a JEM-2100 Transmission Electron Microscope (JEOL Ltd., Japan). 2.6.

Drug loading (DL) and entrapment efficiency (EE)

250 mg of ERY-loaded NDs was added to a size exclusion chromatography column consisting of a syringe 1 ml filled with Sephacryl™S-1000. This procedure allowed separation of the unloaded drug from nanodispersion. The resulting solutions were analyzed by HPLC.

ACCEPTED MANUSCRIPT The drug loading content was the ratio of incorporated drug to lipid (w/w). The DL and EE could be calculated by the following equations:



(%) =

















x 100



x 100

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(%) =

where, Wtotal of encapsulated drug, Wtotal of drug added and Wtotal of lipid were weight of drug encapsulated respectively. The stability of erythromycin in NDs

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2.7.

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into nanoparticles, initial weight of drug in the formulation, and weight of lipid in the system,

Study to determine the stability of API in NDs was carried out in a heated bath at 37±0.5 °C and constant magnetic stirrer (800 rpm). ERY-loaded NDs (4 g) were suspended in 16 g of different media. The media were Simulated Gastric Fluid [SGF - 7.0 ml of HCl, 2.0 g of NaCl, 3.2 g of pepsin (800-2500 U/mg), 1000 ml of Milli-Q water; pH = 1.2 ± 0.05], Simulated Intestinal

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Fluid [SIF - 10.3 g of KH2PO4, 12.4 g of NaCl, 35 ml of 1 N NaOH, 7 g of NaHCO3, 1000 ml of Milli-Q water; pH = 6.8 ± 0.1] and HEPES Buffer [2.4 g of HEPES, 6.3 g of NaCl, 20 mM of sodium citrate, 1000 ml of Milli-Q water; pH = 7.4 ± 0.1]. Each solution was drawn off completely at preset sampling times. The erythromycin solution was then diluted by mobile

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phase and filtered through a 0.20 µm RC-membrane (Minisart, Germany). Drug quantification was performed by HPLC as mentioned above. All assays were done in triplicate. Erythromycin

2.8.

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free-drug was used as a controlled sample. In vitro antimicrobial spectrum study

The minimum inhibitory concentration required to inhibit the growth of 90% of the organisms (MIC) in the ERY-loaded NDs were determined during the stationary phase in an aerated incubator (Mueller Hinton medium). A given amount of erythromycin free-drug, ERY-loaded NDs or unloaded BLANK ND were dispersed in Milli-Q water or SGF with pepsin (pH 1.2) by a magnetic stirrer, and incubated at 37 °C during 6 hours as mother solutions. They were then adjusted by a 1 N NaOH solution to pH 7.5 where erythromycin is more stable

[16]

. Each of the mother solutions was

ACCEPTED MANUSCRIPT diluted by Milli-Q water to 10 samples in a sterile environment. Finally, all of the samples were mixed with Mueller Hinton agar with 10% defibrinated horse blood. The mixture (20 ml) was homogenized and sunk in a sterile Petri plate. Each plate with nutrient agar was inoculated with 1 µL of each microbial suspension (3-4 Mc Farland, indicated by a diode 29 nephelometer of ATP 1550 API). The plates were incubated for 72 hours at 37 °C, and the

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MIC was evaluated for the concentration of antimicrobial agents in culture media ranging from 32 µg/ml to 0.06 µg/ml for the following strains of H. pylori: CCUG 38771, 88-23, CCUG 38772, J99, 84-183. Statistical analysis

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2.9.

The results are presented as mean ± SD (standard deviation). Statistical significance was

3.

Results and discussion

3.1.

Pre-formulation development

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determined using Student’s t-test with p < 0.05 indicating significant difference.

3.1.1. Determination of erythromycin solubilities

The solubility of erythromycin was determined in water and into different excipients. [17]

, the solubility of erythromycin in water is low, approximating

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According to the literature

2.8 mg/g. Concerning erythromycin solubility into lipid excipients, visual examinations were carried out initially. Solubilities of erythromycin in Labrafac®CC, Compritol®HD5ATO,

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Compritol® 88ATO and Labrafil®2125CS were ~6, 8, 10 and 12 wt% respectively. Labrafil®M2125CS being the most interesting excipient, it has been the subject of major

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investigations. Its solubility in lipids, which are viscous fluids, strongly depends on experimental conditions such as temperature or mixing time. To be compatible with a scalable process, mixing time has been set at 4h. The solubility of erythromycin in Labrafil®M2125CS reached approximately 120 mg/g of lipid weight. However, 100 mg/g is the maximum concentration that can be totally dissolved during 4 h; it consequently became the highest concentration used in subsequent experiments.

Erythromycin stability was checked in

®

Labrafil M2125CS at 37 °C. It was totally stable in the lipid phase up to 24 h incubation, confirming that this API is a good candidate for encapsulation. 3.2.

Characterization of the nanoparticles

ACCEPTED MANUSCRIPT 3.2.1. Particle size distribution In this study, Labrafil®M2125CS (20% w/w) as lipid phase and mixture of Gelucire®50/13 – PL90G (2:1) as surfactant were chosen to prepare blank nanodispersions by the hot highpressure homogenization method. The ERY-loaded NDs were formulated by incorporating from 1 to 10% of erythromycin (compared to the lipid phase amount) under the optimized

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processing conditions: 70 °C, 600 bars for 5 min. Drug-free nanodispersions prepared in this study had a mean diameter of 243 ± 3 nm (PdI ~ 0.15). This is higher than that of ERY-loaded NDs, which were approximately 150 nm, and had narrow size distributions (Table 1). The

3.2.2. Drug loading and entrapment efficiency

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PdIs of all samples were below 0.2, indicating distributions homogeneous in size.

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The drug loading of nanoparticles and drug entrapment efficiency (Figure 1) increased from 15 mg/g to 37 mg/g and from 52% to 78%, respectively, with an increase of erythromycin from 3 to 5% (compared to the weight of lipid in formulation). Drug loading was calculated by the API_lipid weight ratio, which reached to 37 mg/g and was constantly kept from 5 to 10% of erythromycin in formulations. It was demonstrated that 5% of erythromycin was the optimal concentration of API that could be loaded into nanodispersions.

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During long-term storage, re-crystallization of API and unpredictable gelation leading decreased o drug entrapment efficiency and drug loading capacity occurred in formulations containing more than 5% of erythromycin.

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The optimal formulation containing 5% of erythromycin, was selected. After purification, optimum entrapment efficiency (EE) and drug loading (DL) were then around 78% and 4% respectively. Drug loading could appear relatively low. However, both values are comparable

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with the EE and DL classically described in the literature for similar lipid-based delivery systems such as nanoemulsions, solid lipid nanoparticles (SLN) or nanostructured lipid carriers (NLC) encapsulating various API

[18]

. Finally, from a physical standpoint, loaded

nanoparticles are stable enough at room temperature. 3.2.3. Cryo-TEM The morphology of ERY-loaded NDs determined by cryo-TEM is shown in Figure 1. The particles had almost spherical shape and did not stick to each other. Their mean diameter was in the range of 150-180 nm. They consisted mainly of nanodroplets and liposomes.

ACCEPTED MANUSCRIPT 3.3.

Stability studies

3.3.1. Stability of ERY-loaded NDs in SGF pH 1.2 In acidic medium, erythromycin A is rapidly degraded via intramolecular dehydration to form erythromycin-6,9-hemiketal and then anhydroerythromycin, both of which possess little antimicrobial activity[17]. The stability of erythromycin in the NDs was monitored (Figure 2).

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Erythromycin free-drug was decomposed at ~ 50% after 6 hours of incubation at 37 °C in SGF with pepsin (pH 1.2), whereas there is no decomposition after encapsulation. 3.3.2. Long-term stability of ERY-loaded NDs

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Table 1 shows the long-term stability of erythromycin-loaded NDs. The mean droplet size of approximately 155 ± 2 nm, the PdI value of less than 0.13 did not change significantly over a

3.4.

Antimicrobial activity studies

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period of 6 months, indicating good resistance against flocculation and coalescence.

A main issue in controlled delivery is to preserve t API integrity and functional activity after the encapsulation process. Erythromycin was encapsulated in the nanodispersions by HPH technique. The present experiment tested on 5 strains of H. pylori to determine ability to

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maintain the antimicrobial activity of erythromycin.

As seen in Table 2, the assays yielded the same MIC values between erythromycin free-drug and encapsulated erythromycin; for 4 strains. On the CCUG 38772, which is resistant to erythromycin, the MICs of ERY-NDs were 4 times lower than the MICs of erythromycin-

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free drugs in water, suggesting a positive role of the formulation. These findings proved that the encapsulation process did not alter erythromycin activity and that the encapsulation of

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erythromycin in nanodispersions provides a desirable therapeutic approach for treatment of H. pylori infection. In vitro anti-H. pylori activity was also investigated in SGF with pepsin (pH 1.2) medium on reference strains of H. pylori (CCUG38771) in view of evaluating the efficacy of the encapsulation. When encapsulated in nanodispersions, MIC of API improved 4-fold on CCUG38771 (Table 2). In acidic conditions, even after an extended incubation period, encapsulated API is more efficient than non-encapsulated API on the sensitive reference strain. Determination of the MICs using the agar dilution technique revealed that lipids used in the formulation inhibited the growth of H. pylori. Indeed, unloaded nanoemulsions also showed antimicrobial activity (Table 2), which was calculated according to lipid amounts.

ACCEPTED MANUSCRIPT Several reports in the literature have focused on lipid activity against H. pylori. It has been found that diets rich in polyunsaturated fatty acids lower the incidence of peptic ulcer and the apparent importance of H. pylori infection in peptic ulcers[15]. In vitro, mono- or polyunsaturated C18 and C20 fatty acids have been shown to be strongly bactericidal against H. pylori by disrupting the bacterial cell membrane[20]. The concentration required to inhibit

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growth by 50% was around 50 µg/ml for both fatty acids. Polysorbate 80, a nonionic surfactant possessing a polyoxyethylene (20) sorbitan head group and a monooleate lipid chain, manifests

bactericidal activity against H. pylori and exerts a synergistic effect with

some APIs, such as clarithromycin. Depending on the pathogen strains, minimal bactericidal concentrations range from 6.6 to 32 µg/ml. Our nanodispersions were composed mainly of

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Labrafil® M2125CS, which contains esters of linoleic acid and polyoxyethylene (6 PEGunits), a compound intermediate between fatty acids and polysorbates. Labrafil® also

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M2125CS contains glycerides , which have been described as bactericidal against H. pylori, particularly monoglycerides[22]. The bactericidal activities measured in vitro for our unloaded lipid-based formulations, from 8 to 32 µg/ml depending on the strains are at once promising and perfectly coherent with the literature.

Unfortunately, a small trial on patients with ulcer showed a less significant in vivo effect of [23]

. Such disappointing results

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fatty acid diets on colonization of the stomach by H. pylori

could be due to the inaccessibility of the bacteria underneath the mucus layer. In the case of fatty acids, their lipophilicity and their potential negative charge in the stomach region where pH is high are strong limitations to their diffusion into the mucus layer[15]. Our formulation

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requires additional discussion with these considerations in mind. The main parameters controlling the diffusion of nanodispersions are their size, surface

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charge and nature. Beyond 200 nm, diffusion of particles through normal gastric mucus dramatically decreases[24].Fortunately, all of our loaded nanodispersions have sizes below this threshold value. Because of the presence of Gelucire® 50/13 as a stabilizer, the nanodroplets are covered by a PEG1500 layer. Lai et al.

[25-27]

discussed the advantages of such PEG-

coatings for the development of mucous-penetrating nanoparticles. In light of their findings, they concluded that coating particles with a high density of short PEG (<2 kDa) chains reduced particle-mucus adhesive interactions: the molecular weight of PEG was too low to support adhesion via polymer interpenetration and the PEG density sufficed to effectively shield the hydrophobic core of the particles, leading to better diffusion properties. Gelucire® 50/13, used in our formulations, contains ester of stearic acid and 1.5 kDa PEG. In addition to

ACCEPTED MANUSCRIPT PEG chain length, another aspect seems favorable to diffusion rather than adhesion. Around 60% of the esters constituting Gelucire® 50/13 are diesters with fatty acids at both terminations of the PEG chain. As they are anchored by both sides in the nanoparticle interface, the PEG chain is less free to interpenetrate with mucus biopolymer. All of our investigations confirm the potential interest of protecting acid-sensitive APIs in

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nanoemulsions for H. pylori treatment. Not only were the excipients used pharmaceutically approved for oral administration, but the homogenization process is widely used on the industrial scale, meaning that our nanotechnology is easily scalable. The next steps of the project should consist in in-vivo experiments and the development of nanoemulsions with

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other APIs.

ACCEPTED MANUSCRIPT Acknowledgments We gratefully acknowledge Mr. Jeffrey Arsham, an American translator, for his rereading and revision of the original English-language manuscript.

1.

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27.

ACCEPTED MANUSCRIPT Table 1. Particle size, polydispersity indices of BLANK or ERY-loaded NDs just after preparation or after 6-month storage at room temperature for the optimal formulation.

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Table 2. MIC measurements (µg/ml) of pure erythromycin, ERY-loaded NDs and BLANK-ND on different strains of H. pylori

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Figure 1. A) Drug loading and entrapment efficiency of erythromycin-loaded nanoemulsions

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(3 to 10% of drug). B) Cryo-TEM observation of the optimal formulation (scale bar: 200 nm).

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Figure 2. Stability of free and encapsulated erythromycin (5%) in SGF (pH 1.2)

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After preparation

After 6 months

Size (nm)

PdI

Size (nm)

PdI

ERY-BLANK

243 ± 3

0.15 ± 0.02

-

-

ERY-ND1

158 ± 1

0.11 ± 0.02

-

-

ERY-ND3

159 ± 2

0.11 ± 0.02

ERY-ND5

157 ± 4

0.12 ± 0.02

ERY-ND7

160 ± 3

0.09 ± 0.02

ERY-ND10

163 ± 3

0.11 ± 0.01

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Formulation

EP

-

155 ± 2

0.09 ± 0.02

-

-

-

-

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Data are represented with mean ± SD (n = 3).

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Water incubation

SGF incubation

88-23

J99

84-182

CCUG38771

CCUG38771

Erythromycin

16

< 0.06

0.125

0.125

0.25

16

ERY- ND5 Lipid concentration

4 800

< 0.06 12.5

0.125 25

0.125 25

0.25 50

4 800

800-1600

1600

1600-6400

1600

1600

1600

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BLANK-ND (MIClipid)

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CCUG38772

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B

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A

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* p < 0.05, for the comparison of erythromycin percentage at start point

ACCEPTED MANUSCRIPT Highlights - Erythromycin loaded nanoemulsion is an efficient system for H. pylori treatment - The nanodispersion improved the stability of erythromycin in an acidic medium. - The encapsulation process did not alter the inhibitory activity of erythromycin.

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- The lipid-based excipients were shown to have a complementary effect.