A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb 5 6

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A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery

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Jin Jau Liau a, Sarah Hook b, Clive A. Prestidge c, Timothy J. Barnes a,⇑

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a

School of Pharmacy and Medical Sciences, Sansom Institute, University of South Australia, Adelaide, SA 5000, Australia School of Pharmacy, University of Otago, Dunedin, New Zealand c Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia b

a r t i c l e

i n f o

Article history: Received 8 January 2015 Revised 27 August 2015 Accepted in revised form 30 September 2015 Available online xxxx Keywords: Liposome Emulsion Oral Multi-compartment Vaccine In vitro

a b s t r a c t The gastric mucosa provides the entry point for the majority of pathogens, as well as being the induction site for protective immunity; however, there remain few examples of oral vaccines due to the challenges presented by the gastrointestinal route. In this study, we develop a lipid-based multi-compartmental system for oral vaccine delivery. Specifically, we have optimised the formulation of a water-in-oil-in-water double emulsion prepared from a triglyceride – soya bean oil, using surfactants Span 80/Tween 80 and Pluronic F127 to stabilise the internal and external water phases, respectively. Into the internal water phase, we also incorporated a PEGylated liposome, prepared using hydrogenated phosphatidyl choline as a carrier for our model protein, FITC–labelled ovalbumin. We demonstrated the successful incorporation of intact liposomes into the internal water phase of the double emulsion using imaging techniques including cryo-SEM and confocal microscopy. Finally, we use in vitro release studies of FITC–ovalbumin, to provide further confirmation of the multi-compartmental structure of the double emulsion system and demonstrate significant extended release of the entrapped model antigen compared with PEG-liposomes; these characteristics are attractive for oral vaccine delivery. Ó 2015 Published by Elsevier B.V.

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1. Introduction

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Vaccination has contributed to the protection of the population from infectious diseases for more than two centuries, progressing from early use of whole cell organisms to the development of peptide subunit vaccines. The use of needles remains the primary administration method for vaccine delivery, which contributes to continuing low patient compliance and high public health costs [1]. However, in recent years, a variety of alternative delivery routes have been investigated, including oral, transcutaneous and intranasal. Microneedles and lipid-based colloidal delivery systems have been used to facilitate transcutaneous immunisation [2]. As a nasal vaccine delivery system, chitosan hydrogel was observed to prolong the residence time of antigen in the nasal cavity and stimulate CD8+ memory cell production [3].

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Abbreviations: FITC, fluorescein isothiocyanate; OVA, ovalbumin; PEG, polyethylene glycol; LP, liposomes; LPDE, liposomes-in-double emulsion; SEM, scanning electron microscope. ⇑ Corresponding author. Tel.: +61 8 83022334; fax: +61 8 83022263. E-mail addresses: [email protected] (J.J. Liau), [email protected]. nz (S. Hook), [email protected] (C.A. Prestidge), [email protected]. au (T.J. Barnes).

Oral delivery of vaccines presents an attractive alternative, yet there are currently no licensed oral sub-unit vaccine products. Current oral vaccines that prevent cholera and typhoid are produced from live attenuated organisms [4]. Protein vaccines, while having the benefit of increased safety, present numerous formulation challenges as they have inherently short half-lives in vivo owing to their susceptibility to both physical and chemical degradation processes [5]. Oral antigen delivery in itself presents two additional challenges, including the presence of enzymes in the gastrointestinal tract as well as the poor permeability and uptake of proteins across the intestinal mucosa. Oral vaccine delivery can be improved with the use of carriers to protect antigen and enhance their uptake by Peyer’s patches [6]. For example fluorescently labelled ovalbumin entrapped in chitosan microparticles was taken up in Peyer’s patches whereas unentrapped ovalbumin was not [7]. Various nanoparticle carriers have been considered for oral vaccine delivery; including solid state delivery systems such as porous silica [8] and poly(lactic-co-glycolic) acid (PLGA) nanoparticles [9]. More recently, there has been growing interest in the use of soft colloidal systems for example, liposomes and emulsions for use as drug [10] and vaccine delivery [11].

http://dx.doi.org/10.1016/j.ejpb.2015.09.018 0939-6411/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: J.J. Liau et al., A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.09.018

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Liposomes have recently found many applications in cancer therapy [12] and as vaccine adjuvants [13], while previously it was shown that polymerised liposomes exhibited potential for use in oral vaccine delivery [14]. Subsequently, liposomes surface coated with polyethylene glycol (PEG) were investigated for oral delivery of insulin and were found to have increased stability when compared to uncoated liposomes in the gastrointestinal tract [15]. Similarly, PEGylated liposomes used for delivering oral recombinant human epidermal growth factor showed an increase in bioavailability and significant gastric ulcer healing effect compared to a protein solution [16]. Alternatively, liposomes stabilised using silica nanoparticles have successfully been prepared for oral insulin delivery [17]. Although liposomes provide potential benefits as an antigen encapsulation system, the challenge remains in protecting the liposome themselves during their transit of the stomach prior to their reaching the intestinal tract. Lipid emulsions stabilised using either surfactants or nanoparticles (e.g. silica) [18] have been extensively studied for parenteral vaccine delivery [19]. However, given that proteins typically require an aqueous environment, a simple oil-in-water emulsion has limited use. In contrast, double emulsions, for example a water-in-oil-in-water (W1/O/W2) emulsion, where W1 and W2 are the internal and external water phases respectively, provide a suitable internal aqueous compartment. Double emulsions have previously been used to encapsulate, control the release and allow the safe passage of bioactives through the gastrointestinal tract in food applications [20]. Double emulsions have also been extended to the field of oral vaccines. A model antigen, ovalbumin was successfully delivered via double emulsions utilising squalene for the oil phase and showed enhancement of both the mucosal IgA and systemic IgG responses [21]. Further refinement of the double emulsion system can be achieved by incorporation of an additional internal compartment (e.g. nanoparticles) to carry and further protect the vaccine. For example, a multi-compartmental system of liposomes-in-single emulsion droplets has been developed for parenteral delivery to treat a variety of diseases including Hepatitis B [22], HIV [23], human papilloma virus (HPV) [24] and melanoma [25]. The liposome-in-emulsion system successfully eradicated tumours in rabbits and created a depot effect thereby leading to the requirement for fewer injections [25]. Recently, the liposomes-in-double emulsion system was adapted for dermal delivery of bovine serum albumin (model protein) and demonstrated controlled antigen release [26]. Furthermore, nanoparticles in emulsions have been investigated for orally delivering nucleic acids and showed effective gene and siRNA delivery [27]. Though the multicompartmental liposomes-in-emulsion system has been applied for parenteral and dermal delivery, such a system has not been investigated for oral subunit vaccine delivery. In this work, we will establish an optimised, stable double emulsion carrier and demonstrate enhanced protection and release of encapsulated antigen containing PEGylated liposomes for oral vaccine delivery. Specifically, we will optimise the surfactant mixture (Span 80: Tween 80) used to stabilise the internal water phase (W1). We will demonstrate the successful incorporation of intact PEGylated liposomes within internal aqueous phase, using both confocal microscopy and cryo-SEM. Finally, using in vitro release studies of FITC–labelled ovalbumin, we will demonstrate the extended release of model antigen provided by the multicompartment liposome-in-double emulsion (LPDE) carrier system.

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2. Materials and methods

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Hydrogenated phosphatidylcholine from soya bean (P90H) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEGDSPE2000) were kindly supplied by Lipoid GmbH, (Germany).

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Cholesterol, chloroform, fluorescein isothiocyanate isomer I (FITC), ovalbumin from chicken egg white, Pluronic F127, Tween 80, Rhodamine B, Coumarin 6, Nile red, phosphate buffered saline (PBS) (10 mM), carbonate-bicarbonate buffer (50 mM) tablets and soya bean oil were purchased from Sigma Aldrich (USA). Span 80 was obtained from Wako Pure Chemical Ind. (Japan).

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2.1. Preparation of liposomes

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Liposomes were prepared via a thin film hydration method [28]. The lipids were dissolved in chloroform and evaporated under a vacuum. The lipid mixture composed of P90H, cholesterol and mPEG-DSPE2000 (molar ratio 55.5:37:7.5). The phospholipid film was hydrated with PBS above the lipid transition temperature (55 °C). The rehydration of the thin film produced a coarse liposome suspension, which was then extruded (Lipex, Canada) through a series of polycarbonate membranes (400, 200 and 100 nm) (WhatmanÒ, USA) to obtain unilamellar liposomes. Fluorescein isothiocyanate (FITC) was conjugated to ovalbumin (OVA) by following a previous method [29]. Liposomes rehydrated with PBS containing 300 lg/mL FITC–OVA were centrifuged (100,000g) at 4 °C for 1 h. The supernatant was diluted in 1% w/v TritonÒ X-100. The fluorescence of FITC–OVA in the supernatant was measured by fluorescence spectrophotometry (Varian Cary Eclipse) at kex = 485 nm, kem = 520. The entrapment efficiency of FITC–OVA in liposomes was determined by subtracting the unentrapped concentration from the initial loaded concentration with reference to the standard curves in the linear range of FITC–OVA prepared on the same day.

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2.2. Preparation of water-in-oil (W1/O), water-in-oil-in-water (W1/O/W2), liposomes-in-single emulsion and liposomes-in-double emulsion

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An extensive formulation optimisation process was undertaken. Double emulsions and liposomes-in-double emulsion were prepared in a two-step emulsification process as described by Wang et al. [26]. In the first step, the internal water phase, W1 was added drop-by-drop to the oil phase with stirring. W1 consisted of hydrophilic surfactant, Tween 80 and PBS or the liposome suspension. The oil phase consisted of lipophilic surfactant, Span 80 and soya bean oil. The water fraction (30–50%) was varied while keeping the surfactant concentrations constant (15.9% v/v Span 80 and 2.8% v/v Tween 80). At a constant 50% water fraction, different volumes (1.25, 6.25, 12.5 and 15.9% v/v) of Span 80 and a constant concentration of Tween 80 at 2.8% v/v were used for the study of varying Span 80 concentration. The water-in-oil (W/O) single emulsion was formed by homogenisation (CAT Unidrive 1000, CAT Scientific, Ca., USA) for 5 min at 3800 rpm. The W2 phase, the polymeric surfactant Pluronic F127 at a concentration of 0.5% w/v in PBS was added to the water-in-oil emulsion (W1/O) and stirred for 30 min at 350 rpm to form the double emulsions (W1/O/W2). Various volumetric ratios of W1:O:W2 of the double emulsion were also investigated.

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2.3. Particle size and zeta potential

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The size distribution and zeta potential of the liposomes were measured using a Zetasizer Nano-ZS (Malvern Instruments, UK). The particle size distribution of the double emulsions was determined by laser diffraction (Mastersizer 2000, Malvern, UK).

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2.4. Cryo-SEM

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SEM imaging of water-in-oil emulsion (W/O), water-in-oil-inwater (W/O/W) emulsion and liposomes-in-double emulsion was

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Please cite this article in press as: J.J. Liau et al., A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.09.018

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carried out as described previously by using Field Emission SEM (Philips XL30) and cryostage (Gatan CT1500 HF) [26]. Images were acquired at a voltage of 5 kV.

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2.5. Fluorescent labelling and confocal fluorescence microscopy

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The emulsions were visualised using a confocal microscope (A1-R, Nikon) with 60
objective and analysed using NIS elements AR software. The lipid dye, Coumarin 6 (0.3 mol%) was added to the chloroform phospholipid solution while the aqueous dye, Rhodamine B (0.1 mM) was added during the rehydration of the thin film followed by the removal of excess Rhodamine dye by centrifugation, decanting the supernatant and resuspending the liposome pellet in PBS. The dye-labelled liposomes were then encapsulated in the internal water phase, W1 of the double emulsion. In another imaging study to visualise the entrapment of FITC–OVA in the system, the oil phase was labelled with lipid dye (6 lg/mL Nile red) while liposomes were loaded with 200 lg/mL FITC–OVA during rehydration of the thin film. The excess FITC–OVA was removed by centrifugation as described above. The dye and fluorescent-labelled emulsions were transferred to a glass slide before a drop of water was added and a cover slip applied for imaging.

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2.6. In vitro release studies

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The release of FITC–OVA from the liposomes and double emulsions was determined using a modified dialysis bag method [30]. Liposomes, double emulsions and liposomes-in-double emulsion (liposomes contained entrapped and unentrapped FITC–OVA) were added into a 100 kDa cellulose ester membrane dialysis bag (Spectra/PorÒ, Spectrum Laboratories Inc., USA) which was soaked for 30 min in water and rinsed thoroughly before use. The formulation loaded dialysis bag was then immersed in a dissolution media (0.01 M PBS, pH 7.4) contained in a beaker and maintained at 37 °C in a water bath. The dissolution media was stirred continuously during the 8 h study. At specific intervals, aliquot samples were taken from the dissolution medium and replenished with PBS. The samples were analysed using fluorescence spectrophotometry for FITC–OVA quantification.

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Span (%) Fig. 1. Single (W1/O) emulsion droplet size, influence of (A) water fraction and (B) Span concentration. Error bars show standard deviation from triplicate measurement.

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3. Results

remaining W1/O emulsions exhibited varying degrees of stability over 1 week, as shown in Fig. 2A. The most stable single emulsions (W1/O) were stabilised by the highest concentration of lipophilic surfactant (Span 80; 15.9%) investigated. W1/O emulsions prepared from Span 80 (15.9% v/v), Tween 80 (2.8% v/v) and 50% water fraction were considered optimal and used for the following studies.

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3.1. Preparation and optimisation of the internal W1/O emulsion

3.2. Double W1/O/W2 emulsions: Characterisation and stability

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The internal water phase (W1) was stabilised using two surfactants, the lipophilic Span 80 and the hydrophilic Tween 80. The influence and role of both water volume fraction (30–50%) and Span 80 concentration (1.25, 6.25, 12.5 and 15.9% v/v) were investigated to optimise the stability of the emulsion while the Tween 80 concentration was kept constant (2.8% v/v). Using a fixed Span 80 (15.9% v/v) and Tween 80 (2.8% v/v) concentration ratio, and increasing the water (W1) fraction from 30% to 50%, we observed a decrease in the average droplet diameter, from 1300 to 900 nm (Fig. 1A). Next, we varied the Span 80:Tween 80 ratio, by varying the Span 80 concentration from 15.9% to 1.25% v/v (constant concentration Tween 80). This decrease in Span concentration resulted in an increase in the effective HLB from 6 to 12. Above 5% Span 80, droplets of approximately 700–900 nm in diameter were observed (Fig. 1B). The influence of Span 80 concentration on the macroscopic physical stability of the W1/O emulsion was assessed by visual inspection (Fig. 2), with the emulsions observed to be stable for more than 24 h. After 24 h, visible phase separation was observed with the lowest Span 80 concentration (1.25%); however, the

The double emulsion formulation was optimised with respect to the internal water volume fraction, and (W1:O:W2) volumetric ratios of 1:1:4, 1:1:2, 1:1:1 and 2:2:1 were investigated. In general the double emulsions exhibited good stability, with only limited phase separation were observed after storage for 1 week (Fig. 2B). From our observations, the most stable double emulsion system was determined to be composed of 36% W1 phase, 44% oil phase and 20% W2 phase. The lipid droplets of the double emulsions and liposomes-indouble emulsion displayed a bimodal volume droplet size distribution with droplet size peaks at 700 nm and 5 lm (Fig. 3). This was not unexpected, given the relatively low shear used to prepare the double emulsion system, in order to preserve the internal water (W1) phase. The cryo-SEM images of the emulsions (W/O and W/O/W) and liposomes-in-double emulsion are shown in Fig. 4. The indentations in the single water-in-oil (W/O) emulsion image indicate water droplets (Fig. 4A). The image of the double emulsion showed a droplet within a droplet, indicative of a double emulsion (Fig. 4B). Upon addition of liposomes into the internal aqueous phase, W1,

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Size (µm) Fig. 3. Representative droplet size distribution data of double emulsions (j) and liposomes-in-double emulsions (d).

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Water Oil

Fig. 2. (A) Visualisation of single (W1/O) emulsions, and the influence of Span 80 concentration (from left to right: 1.25, 6.25, 12.5, 15.9% v/v); after 1 week. (B) Visualisation of double (W1/O/W2) emulsions after 1 week, with various W1:O:W2 ratios (from left to right; 1:1:4, 1:1:2, 1:1:1 and 2:2:1).

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the external surface of the lipid droplets visualised appeared rough (Fig. 5A). Upon close observation of the liposomes-in-double emulsion, small indentations could be seen within the internal aqueous phase, W1 which were attributed to liposomes (Fig. 5C) in agreement with previous observations reported in the literature [26].

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3.3. Preparation and characterisation of OVA-liposomes-in-double emulsions The diameter of the PEGylated liposomes was 150 to 170 nm with low dispersity (PDI 0.1), in the absence and presence of the encapsulated ovalbumin model antigen. The liposomes were negatively charged with a zeta potential of 22 ± 4 mV; this did not change upon loading with FITC–OVA. The entrapment efficiency of FITC–OVA was 43 ± 10% and no further purification of the OVA entrapped liposomes was performed. In this study, confocal microscopy was used to visualise the location of the nanocarriers within the double emulsions. Liposomes were labelled with a red aqueous dye, Rhodamine B and green lipid dye, Coumarin 6 prior to encapsulation in the internal water phase (W1) of the double emulsions. The aqueous dye, Rhodamine B and lipid dye, Coumarin 6 appear to locate within the oil droplet (Fig. 6A). There were typically 1–4 water droplets within each oil droplet. The size of the oil droplets was in good agreement with the results obtained for the W1/O single emulsion system. Finally, we characterised our multi-compartmental (W1/O/W2) vaccine carrier in the presence of OVA-liposomes within the

5 µm Fig. 4. Cryo-SEM image of (A) single emulsions (W/O) and (B) double emulsions (W/O/W).

internal (W1) phase. We again used confocal microscopy to directly ascertain the successful incorporation of the hydrophilic antigen, FITC–OVA in liposomes using confocal microscopy (Fig. 6B). FITC–OVA was visualised within the double emulsions. The lipid dye, Nile red, when added to the oil phase labelled the lipid droplets red. The presence of liposomes in the multi-compartmental system was confirmed.

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3.4. In vitro OVA release studies from nanostructured carriers

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The release kinetics of FITC–OVA from liposomes, double emulsions and liposomes-in-double emulsion is presented in Fig. 7A.

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The in vitro release of OVA from liposomes containing both entrapped and unentrapped FITC–OVA exhibited a biphasic release profile showing rapid release in the first hour followed by a more gradual release after 2 h. The initial rapid release is likely from the unentrapped OVA within W1 of the formulation. The double emulsions and liposomes-in-double emulsion exhibited more sustained release compared to liposomes. In the first hour, liposomes released 35% of OVA as opposed to 11% and 15% from double emulsions and liposomes-in-double emulsion respectively. Liposomes encapsulated in double emulsions retarded OVA release enhancing the sustain release of antigens in vitro. Ovalbumin release kinetic data were further analysed using the Peppas model, which is defined by the following equation:

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Mt ¼ atn M1

Fig. 5. (A) Cryo-SEM images of liposomes-in-double emulsion (LPDE). (B) Close observation of the cross-section of LPDE (B) and liposomes as indicated by arrows within W1 droplet (C).

ð1Þ

where t is time, Mt/M1 is the fraction of antigen released, a is a constant incorporating structural and geometrical characteristics of the formulation and n is the release exponent. The linearised release data for liposomes, double emulsions and liposomes-in-double emulsion are presented in Fig. 7B. The linearised OVA release data for the liposomes, double emulsions and liposomes-in-double emulsions showed good linearity (Fig. 7B and Table 1), indicating the suitability of the Peppas model. Ritger and Peppas [31] introduced the release exponent limits for the interpretation of solutes released from spheres. The release exponent (n) obtained from the Peppas model ranged from 0.33 for liposomes to 0.70 for the double emulsions. The release exponent provides insight into the mechanism of OVA release, for example, the OVA release from liposomes could be described by a Fickian diffusion process, given n < 0.43. In contrast, OVA release from double emulsions and liposomes-in-double emulsion was described as a non-Fickian diffusion process, as 0.43 < n < 0.85, indicating the involvement of more than one release mechanism. This is not surprising given the multiple physical barriers to the release of OVA from the multiple emulsion systems.

Fig. 6. Confocal images of liposomes-in-double emulsion; (A) liposomes incorporated with lipid dye, Coumarin 6 (green) in lipid bilayer and loaded with Rhodamine B (red) in the aqueous core. (B) Oil layer dyed with Nile red (red) and liposomes loaded with FITC–OVA (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: J.J. Liau et al., A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.09.018

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OVA release (%)

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Fig. 7. (A) Release of FITC–OVA from liposomes (s), liposomes-in-double emulsion (h) and double emulsion (4) and (B) release kinetics fitted using the Peppas model. Release data are the mean ± SD of at least three independent experiments.

formulations and as it consists of long chain triglycerides, and digestion by lipase is prolonged [35]. The long chain lipids will reduce the digestion rate of the multiple emulsion carrier [36], thereby controlling the release of OVA/OVA-in-liposomes from the multiple compartmental system during gastric transit. This in turn increases the likelihood of delivering the OVA undamaged/ bioactive to access Peyer’s patches within the intestines. The stability of double emulsions is complex, depending on the particle size of the inner (water) and outer (oil) droplets, which in turn are governed by a number of factors including the type and concentration of emulsifiers, interfacial tension and phase volume fraction [37]. The most stable water-in-oil emulsion was prepared with a HLB value of 6 which falls in the HLB range (2–7) reported to form stable water-in-oil emulsions [38]. The stable double emulsion formed with high ratio of Span 80 to Tween 80 at the W1/O interface is in agreement with the observation of Hou and Papadopoulos [39]. Studies have shown that high concentrations of lipophilic surfactant increase the rigidity of the oil globule of the double emulsion hence, reducing the swelling rate of the internal water phase [40] and coalescence between oil droplets [41].

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4.3. In vitro release behaviour

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The release of OVA from double emulsion and liposomes-indouble emulsion formulations are significantly more complex than for liposomes alone. In vitro OVA release from liposomes follows Fickian diffusion as opposed to the double emulsion formulations’ non-Fickian diffusion mechanism, where surfactants are required to facilitate transport from the internal (W1/O) to the external interface (O/W2) [42]. The transport mechanism across the oil phase is most likely due to diffusion via reverse micelles [43]. Therefore, hydrophilic antigens such as OVA diffuse from the internal water phase, W1 to the external water phase, W2 by being incorporated into reverse micelles formed by lipophilic surfactant, ie. Span 80. In addition, water transport could also occur when the internal water droplet, W1 contacted the external aqueous phase, W2 [44].

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5. Conclusion

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4. Discussion

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4.1. Surface modification of liposomes

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The liposomes were PEGylated for increased stability in the gastrointestinal tract, as previously described by Iwanaga et al. [15]. Increasing liposome PEGylation which was correlated with a thicker layer of lipid bilayer has been previously found to reduce the release of the entrapped model hydrophilic drug [32]. The release of OVA from the PEGylated liposomes was hindered due to the PEG on the surface of the liposomes, which also provides a steric barrier to stabilise the liposomes within the internal water phase of the double emulsion. The attachment of the PEG (7.5 mol%) on to the surface of the liposomes induced stability by increasing repulsive forces (steric, electrostatic and hydration) and reducing aggregation [33].

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4.2. Stability of double emulsions

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Lipid-based systems, liposomes and emulsions have been widely used for the delivery of antigens due to their biocompatibility [34]. Similarly, soya bean oil is regularly used in pharma

We have successfully developed a multi-compartmental system protein antigen-carrier system, based on liposome-in-water-in-oilin-water (W1/O/W2) double emulsions (LPDE). We have optimised the Span/Tween surfactant ratio and water volume fraction to prepare a stable double (triglyceride) emulsion system. We demonstrated the successful incorporation of OVA-loaded liposomes into the internal water (W1) phase of the double emulsion, by direct visualisation using both confocal and cryo-SEM imaging. Finally, using in vitro release studies of a model antigen, FITC– labelled ovalbumin, we provide further confirmation of the multi-compartmental structure of the double emulsion system and demonstrate significant extended release of the entrapped model antigen compared with PEG-liposomes. This multicompartmental LPDE system offers significant potential as an oral vaccine delivery system.

Table 1 Peppas model fitting parameter to OVA release from different formulations.

Acknowledgements

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The authors would like to thank Haley Scarborough and Michelle Bassett for assisting with the in vitro studies. We would also like to acknowledge Lynette Waterhouse at the Adelaide Microscopy, University of Adelaide for the assistance with the cryo-SEM imaging of the emulsions.

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Formulations

Liposomes Double emulsions Liposomes-in-double emulsion

Peppas model n

R2

Release mechanism

0.33 0.70 0.56

0.974 0.993 0.995

Fickian diffusion Non-Fickian diffusion Non-Fickian diffusion

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Please cite this article in press as: J.J. Liau et al., A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.09.018

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