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Solid-in-oil nanodispersions for intranasal vaccination: Enhancement of mucosal and systemic immune responses
International Journal of Pharmaceutics 572 (2019) 118777
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Solid-in-oil nanodispersions for intranasal vaccination: Enhancement of mucosal and systemic immune responses
T
Qingliang Konga, Momoko Kitaokaa, Yoshiro Taharaa, Rie Wakabayashia,b, Noriho Kamiyaa,b,c, ⁎ Masahiro Gotoa,b,c, a
Department of Applied Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan Advanced Transdermal Drug Delivery Center, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan c Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Immunization Intranasal vaccination Solid-in-oil nanodispersion Vaccine
En masse vaccination is a promising strategy for combatting infectious diseases. Intranasal vaccination is a viable route of mass vaccination, and it could be performed easily via needle-free administration. However, it is not widely used because it tends not to evoke sufficient immunity. The aim of the present study was to improve the performance of intranasal vaccination by extending the amount of time that administered antigens remain in the nasal cavity, and enhancing immune responses via a nanocarrier-based adjuvant. A simple and safe solid-in-oil (S/O) system was investigated as a nanocarrier in intranasal vaccination. S/O nanodispersions are oil-based dispersions of antigens coated with surfactants. Because of the mucoadhesive capacities of surfactant and oil they have high potential to extend the amount of time that administered antigens remain in the nasal cavity, and can induce strong immune responses due to a nanocarrier-based adjuvant effect. In nasal absorption experiments antigens administered intranasally via S/O nanodispersions remained in the nasal cavity longer and induced strong mucosal and systemic immune responses. Histopathology analysis indicated that S/O nanodispersions did not modify the nasal epithelium or cilia, suggesting non-toxicity of the carrier. These results indicate the potential of intranasal vaccination using S/O nanodispersions for future vaccination.
1. Introduction Frequent outbreaks and rapid spread of airborne infectious diseases such as influenza, tuberculosis, and measles are major public health problems worldwide. Vaccination is the most effective method for protecting against these infectious diseases. The common methods of vaccination via subcutaneous, intradermal, and intramuscular injection have several drawbacks however, such as low patient compliance, need for administration by a medical professional and needle-stick injuries (Narwaney et al., 2017; Trinh et al., 2017). Moreover, mass vaccination via injection is problematic after an infectious diseases has already broken out and spread worldwide, particularly in developing countries (Levine, 2003). In addition, injection can induce protective systemic antibodies but does not induce substantial mucosal antibodies, which contribute to the prevention of infectious diseases (Lycke, 2012). To overcome these issues, more suitable and effective vaccination
strategies are urgently needed. Intranasal vaccination has recently emerged as an attractive alternative to subcutaneous, intradermal, and intramuscular vaccination. Because intranasal vaccination is needle-free and non-invasive, it could be used for large-scale immunization without an inherent need for a trained medical professional. The nasal mucosa is easily accessible and highly vascularized, and has abundant immune cells with the potential to induce effective immune responses. In addition, nasal administration is reportedly able to induce both systemic and mucosal immune responses (Lycke, 2012). Since most infections start from mucosal surfaces, mucosal immunity is important as a first-line of pathogen defense. The pathogen-specific antibodies are stimulated by mucosal vaccination and secreted into the mucus to neutralize the pathogen. The main mechanism of the induction of immunity via intranasal vaccination is that antigens in the vaccine are transported via microfold cells in the nasal epithelium, then resident dendritic cells capture the antigens,
Abbreviations: ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein-4-isothiocyanate; H&E, hematoxylin and eosin; Ig, immunoglobulin; IPA, isopropanol; IVIS, in vivo imaging system; L-195, sucrose laurate; OD, optical density; OVA, ovalbumin; O/W, oil-in-water; PBS, phosphate-buffered saline; S/O, solid-in-oil; TEM, transmission electron microscopy; W/O, water-in-oil; W/O/W, water-in-oil-in-water ⁎ Corresponding author at: Department of Applied Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan. E-mail address: [email protected] (M. Goto). https://doi.org/10.1016/j.ijpharm.2019.118777 Received 8 June 2019; Received in revised form 29 September 2019; Accepted 7 October 2019 Available online 31 October 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.
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(Osaka, Japan). Perilla oil was purchased from Kaneda (Tokyo, Japan). A surfactant sucrose laurate (L-195) was kindly provided by MitsubishiKagaku Foods (Tokyo, Japan). Horseradish peroxidase-labeled rabbit anti-mouse Ig (immunoglobulin) G, IgG1, and IgG2a were obtained from Rockland Immunochemicals (Gilbertsville, PA, USA). All other reagents used were of analytical grade. Six-week-old female BALB/c mice were purchased from Kyudo, Co. (Saga, Japan) a week prior to experimentation, and maintained under standard conditions. All mouse experiments were approved by the Ethics Committee for Animal Experiments of Kyushu University, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Science Council of Japan).
process them, and induce immune responses (Marasini, 2017; Shakya et al., 2016; Zaman et al., 2013). Despite these advantages, only a few commercially available intranasal vaccines, such as Flumist®, Fluenz®, and Nasovac®, have been developed (Lycke, 2012). The main challenges include degradation of free antigen in the vaccine by enzymes in the nasal cavity, the short time that antigens remain in the nasal cavity due to rapid nasal mucociliary clearance, and weak immune responses resulting from inefficient antigen transportation to the immune system (Marasini, 2017). Therefore, the development of an effective vaccine formulation that can maintain antigen stability, extend the amount time that antigen stays in the nasal cavity, and stimulate immune responses as an adjuvant is required. To date, nanogel (Fukuyama et al., 2015; Nochi et al., 2010), liposomes (Ghaffar et al., 2016; Tada et al., 2015), emulsions (Makidon et al., 2012; Wong et al., 2014), lipid (Battaglia et al., 2018), and polymeric nanoparticles (Lebre et al., 2016; Pawar and Jaganathan, 2016) have been developed as effective formulations for intranasal vaccination. Of these, emulsions have attracted much attention due to the ease of manufacturing them and scaling up production, and the adjuvant effect caused by constituent surfactants and oils (Fox, 2009; Wong et al., 2014). Notably however, oil-in-water (O/W) emulsions—which are the most common emulsions used in intranasal vaccines—have several drawbacks including the short amount of time that antigen remains in the nasal cavity due to the low viscosity of waterbased emulsions, and low loading efficiency of antigens because they are simply mixed with oil-droplets and not encapsulated inside oil. In some cases, bursting or incomplete release causes only part of the antigen to be transported to the immune system (Aucouturier et al., 2001; Mahajan and Rasal, 2013). In an effort to overcome these issues, intranasal vaccination involving a solid-in-oil (S/O) nanodispersion technique was investigated in the present study. S/O nanodispersions are oil-based dispersions containing nano-sized particles consisting of antigen coated by hydrophobic surfactant molecules (Kitaoka et al., 2016). In contrast to waterbased emulsions such as O/W and water-in-O/W (W/O/W) emulsions which have traditionally been used in intranasal vaccination, oil-based S/O nanodispersions exhibit high stability for more than 3 months due to reduced opportunity for Ostwald ripening (Kitaoka et al., 2016), and high antigen encapsulation efficiency of up to 99.9% (Wakabayashi et al., 2018). Moreover, as well as extending the amount of time that antigen remains in the nasal cavity such that it is transported to the immune system, the oil-based formulation of S/O nanodispersions can reportedly act as an adjuvant and strengthen immune responses (Fox, 2009). To the best of our knowledge few oil-based formulations have been developed for intranasal vaccination (Bonferoni et al., 2019), and only the delivery of insulin via the nasal route using water-in-oil (W/O) microemulsions has been reported (Sintov et al., 2010). In the current study the potential of intranasal vaccination using S/ O nanodispersions loaded with the model antigen ovalbumin (OVA) was investigated. S/O nanodispersions were prepared using different oils, and formulation optimization was attempted. Nasal absorption and the amount of time that OVA remained in the nasal cavity after administration in an S/O nanodispersion were assessed. S/O nanodispersions were administered to mice intranasally for the evaluation of OVA-specific immune responses. Lastly, the health of the nasal mucosa was assessed after intranasal administration of S/O nanodispersions.
2.2. Preparation of S/O nanodispersions in different oils S/O nanodispersions were prepared as previously described (Kitaoka et al., 2016). A W/O emulsion was prepared from an aqueous OVA solution (0.5 mg/mL) and a cyclohexane solution of surfactant L195 (12.5 mg/mL) using a PT2500E polytron homogenizer (Kinematica AG, Luzern, Switzerland) at 26,000 rpm for 2 min. The W/O emulsion was flash-frozen in liquid nitrogen for 20 min then lyophilized for 24 h with an FDU-1200 lyophilizer (Eyela, Tokyo, Japan). Lastly, the resulting viscous surfactant-OVA complex was dispersed in squalane, linseed, perilla, or soybean oil to yield the S/O nanodispersions (Fig. S1). The size distribution of the S/O nanodispersions was analyzed with a Zetasizer Nano ZS light scattering instrument (Malvern, Worcestershire, UK). The viscosity of the S/O nanodispersions was measured with a microviscometer (Lovis 2000 M/ME, Anton Paar, Austria). Morphological analysis was performed by transmission electron microscopy (TEM) using a JEM-2010 (JEOL, Tokyo, Japan). Briefly, the specimen was stained with 2% uranyl acetate, dried in vacuo, and imaged at an accelerating voltage of 120 kV. 2.3. Drug release test Drug release tests were performed using custom-fabricated Franztype diffusion cells with an effective diffusion area of 0.785 cm2 and a receptor volume of 5 mL. A polycarbonate film (Whatman Nuclepore Track-Etch Membrane, 0.1 µm; GE Healthcare) was set on a cell, and the receptor compartment was filled with PBS pH 6.4 which is within the normal murine nasal cavity pH range (Shah et al., 2017). S/O nanodispersions carrying 1 mg/mL of FITC-labeled OVA dispersed in different oils (0.25 mL) were added to the donor phase of Franz-type diffusion cell and the cell was incubated for 6 h at 34 °C, which is within the normal murine nasal cavity temperature range. Samples were extracted from the receptor compartment at 1, 3, and 6 h, and replaced with the same volume of fresh media. FITC-labeled OVA concentrations were measured with a fluorescence spectrometer LS-55 (PerkinElmer, Waltham, MA, USA). 2.4. In vivo fluorescence imaging Mice under anesthesia were intranasally administered 10 μL of PBS solution or S/O nanodispersions containing Cy5-OVA. After 0.5 h, 1.0 h, 2.0 h, and 4.0 h mice were scanned using an in vivo imaging system (IVIS-Lumina II-FS-T3 Xenogen, USA) at an excitation wavelength of 640 nm. Mice administered PBS solution alone were used as controls.
2. Materials and methods 2.5. In vivo nasal absorption 2.1. Materials To investigate the amount of Cy5-OVA adhered to nasal tissue, the nasal cavities of mice were isolated at 0.5 and 2.0 h after intranasal administration of Cy5-OVA PBS solution or S/O nanodispersions. The nasal surface was then washed with an extraction solution consisting of a mixture of PBS, methanol, and acetonitrile (2:1:1 v/v/v) to remove Cy5-OVA from the nasal surface. The nasal tissue was then cut into
OVA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein-4-isothiocyanate (FITC) was purchased from Dojindo (Kumamoto, Japan), and Cy5 mono-reactive dye was purchased from GE Healthcare (UK). Cyclohexane, linseed oil, soybean oil, and squalane oil were obtained from Fujifilm Wako Pure Chemical Corporation 2
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pieces and Cy5-OVA was extracted with extraction solution for 24 h. The cumulative amounts of Cy5-OVA were calculated based on fluorescence intensities measured via the LS55 fluorescence spectrometer.
Table 1 Characterization of solid-in-oil nanodispersions in different oils. S/O in different oils
Diameter (nm)
PDI
Viscosity (mPa s, 25 °C)
Viscosity (mPa s, 34 °C)
Squalane Linseed Soybean Perilla
148.4 ± 6 327 ± 15 958 ± 20 1234 ± 111
0.170–0.241 0.125–0.298 0.044–0.169 0.280–0.348
31.36 38.76 55.58 46.99
21.17 28.30 38.66 32.79
2.6. Murine intranasal immunization Mice were randomly divided into two groups and immunized with OVA-PBS or S/O nanodispersions (10 μL each, 2 mg/mL) once per week for 3 weeks. Mice were held upright immediately after vaccination, to ensure maximal dosing and prevent swallowing. Blood samples were collected from a tail vein 7 days after the third immunization. All mice were then killed via neck dislocation, and nasal mucosal washes were collected through the trachea toward the nasal cavity with 0.5 mL of PBS (Puchta et al., 2014). 2.7. Measurement of serum and mucosal wash antibody levels Serum from immunized mice was assayed via the enzyme-linked immunosorbent assay (ELISA) to determine levels of OVA-specific antibodies (total IgG, IgG1, IgG2a, and IgA) as previously described (Tahara et al., 2010). The antibody titer was defined as the dilution factor at which the optical density of the sample was equal to that of a non-immunized sample. OVA-specific IgA levels in mucosal washes were assayed via ELISA using the Mouse IgA Ready-SET-Go kit (eBioscience, San Diego, CA, USA). Optical density (OD) was read at wavelengths of 450 nm and 570 nm on an iMark microplate reader (Bio-Rad, Berkeley, CA, USA).
Fig. 1. Cumulative release (%) of ovalbumin (OVA) from solid-in-oil nanodispersions into aqueous media. Each point represents the mean ± standard deviation of four replicates. The data reflect percentages of the cumulative amounts of OVA in the receptor chambers.
2.8. Histopathology 34 °C. However, the viscosity of S/O nanodispersions was increased more than 30 times compared with PBS solution. S/O nanodispersions in perilla and soybean oil exhibited the highest viscosity, but S/O in squalane oil exhibited comparatively low viscosity (Table 1). The OVA release efficiency of S/O nanodispersions was evaluated using a Franz diffusion cell instrument. For antigen to be effective it must be released from S/O nanodispersions. Complete release of the OVA was observed within 6 h for the S/O dispersed in squalane oil (Fig. 1). Conversely, less than 15% of the OVA was released from the S/ O dispersed in other oils within 6 h.
The nasal toxicity of S/O nanodispersions was evaluated via hematoxylin and eosin (H&E) staining. Mice received S/O nanodispersions (10 μL) via intranasal instillation. OVA PBS solution was used as a positive control and OVA isopropanol (IPA) solution was used as a negative control (Khunt et al., 2017). At 1 h after administration the mice were killed via an overdose of anesthetic, and the cranium was detached from the vertebrae. The cranium was fixed with 4% paraformaldehyde for 24 h. The nasal cavities of the mice were carefully excised, decalcified with 10% formic acid for 12 h, embedded in optimal cutting temperature compound, then stained with H&E and observed using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) (Hall et al., 2012).
3.2. Nasal absorption of antigen To examine the amount of time that OVA in S/O nanodispersions remained in the nasal cavity, in vivo nasal absorption of Cy5-OVA in mice was evaluated using an IVIS. Mice were intranasally administered the Cy5-OVA in a PBS solution or in S/O nanodispersions, and views of living animals were recorded at predetermined time-points (Fig. 2a). When Cy5-OVA in PBS was administered there was rapid clearance of Cy5-OVA at the site of administration, and almost no fluorescent signal was detected in the nose even at 1 h. However, strong fluorescence was detected in the noses of mice treated with Cy5-OVA S/O nanodispersion, and some signal was detected even at 4 h. To assess differences in amounts of Cy5-OVA adhered to the nasal mucosa, sections of nasal cavities were imaged with a confocal laser scanning microscope. The fluorescent signal in mice treated with S/O nanodispersions was stronger than that of mice treated with PBS solution at 0.5 h, 2.0 h, and 4.0 h (Fig. S4). The amounts of Cy5-OVA remaining in the nasal cavity after vaccination were quantified. At 0.5 h after vaccination S/O nanodispersions exhibited the mucosal adhesion of more than twice the amount of OVA than the PBS solution, and at 2.0 h it was more than four times the amount (Fig. 2b). Collectively the results indicated that S/O nanodispersions prolonged the intranasal retention of OVA in the nasal cavity.
2.9. Statistical analysis The data were expressed as means ± the standard deviation. The ttest was used in statistical analysis, which was performed using GraphPad Prism software. 3. Results 3.1. Characterization of the S/O nanodispersions in the different oils The physicochemical characterization of S/O nanodispersions can be defined by various parameters such as physical appearance, particle size, viscosity, and release efficiency. The physical appearance of S/O nanodispersions containing OVA was transparent and homogeneous in the different oils (Fig. S2). Morphological investigation using TEM suggested that spherical structure of the surfactant-peptide complex was formed (Fig. S3). The size distribution of S/O nanodispersions as measured via dynamic light scattering suggested that the respective mean diameters were 148, 327, and more than 1000 nm in squalane oil, linseed oil, and perilla and soybean oil S/O nanodispersions (Table 1). The viscosity of S/O nanodispersions in different oils was measured using a microviscometer at 25 °C and 34 °C. The OVA in PBS solution exhibited low viscosities of 0.907 mPa s at 25 °C and 0.753 mPa s at 3
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a
Control
PBS sol.
response it also increases local mucosal immune responses, which contribute to blocking initial infection and prevent further infection. Due to the poor immunogenicity of free antigens in intranasal vaccines however, more effective vaccine formulations are required. Nanoparticle systems are commonly used to improve the immunogenicity of antigens because they can facilitate the efficient transport of those antigens to the immune system (Marasini, 2017). Among nanoparticle systems, emulsions—particularly O/W nanoemulsions—have attracted much attention due to the ease of manufacturing and scaling up, and the adjuvanting effects of surfactants and oils (Fox, 2009; Wong et al., 2014). S/O nanodispersions have been developed from W/O emulsions as an oil-based dispersion of nano-sized particles. They are prepared by removing water from a W/O emulsion via lyophilization, and redispersing the surfactant/antigen complex in another oil vehicle. Compared with conventional O/W emulsions, S/O nanodispersions exhibit higher stability and higher antigen encapsulation efficiency. We have previously assessed immune responses to transcutaneous immunization with S/O nanodispersions loaded with OVA (Kitaoka et al., 2014), and more recently we developed pollinosis and cancer immunotherapies using S/O nanodispersions (Kong et al., 2017; Wakabayashi et al., 2018). In our previous studies S/O nanodispersions with an entirely oil base had high viscosity and strong adjuvanting effects compared with free antigen in PBS. Moreover, high viscosity and adjuvant effects are considered essential for intranasal vaccines, which require high mucoadhesive ability in order to ensure that the amount of time antigen remains in the nasal cavity is sufficient to facilitate transport to the immune system, and adjuvanting to strengthen immune responses. Therefore, we hypothesized that S/O nanodispersions may be useful for intranasal vaccination. One crucial aspect of the formulation of S/O nanodispersions for intranasal vaccination is the choice of oil, which determines the size, mucoadhesive capacity, and adjuvanting effect of the formulation. Highly purified non-mineral oils are reportedly well tolerated because they are rapidly metabolized and eliminated (Aucouturier et al., 2001). Squalane oil was deemed to be the optimal oil for the formulation in the present study due to nano-sized particle formation, comparable viscosity, and complete release of antigen. S/O dispersions using squalane oil exhibited a nano-level mean diameter, whereas those using perilla and soybean oil exhibited micro-level mean diameters (Table 1). Nanosized vaccines are preferable for intranasal vaccination because they are associated with more efficient transport of antigen to the immune system (Marasini, 2017). The viscosities of the formulations investigated were evaluated as a potential indicator of mucoadhesive capacity. The viscosities of the S/O nanodispersions were at least 30 times greater than that of the PBS solution, which exhibited viscosity of 0.907 mPa s at 25 °C and 0.753 mPa s at 34 °C (Table 1). Different from water-based chitosan nanoparticles and cationic liposomes, their positive charge binding to the negatively charged mucus (Jabbal-Gill et al., 2012; Tada et al., 2015), the greater viscosity of S/O nanodispersions could promote adhesion to the nasal mucosal surface. Complete release of OVA from S/O nanodispersions in squalane oil occurred within 6 h, while the amount of OVA dispersed in other oils released within 6 h was less than 15% (Fig. 1). Considering the possibility of incomplete antigen release and the rapid clearance of antigen in the intranasal cavity (Riese et al., 2014), complete release within 6 h was considered suitable for an intranasal vaccination. There are several reports of the formulation of stable emulsions based on squalene oil in clinic studies, and in its hydrogenated form it has been preferred due to its high chemical stability, resistance to oxidation, and metabolizing properties (Fox, 2009; Kantipakala et al., 2019). Mechanisms of transdermal antigen delivery via S/O nanodispersions have also been investigated in a previous study (Kitaoka et al., 2014). The normal nasal mucociliary clearance time is only approximately 20 min in humans (Soane et al., 1999). Therefore each intranasal vaccine formulation’s capacity for mucoadhesion, which could slow down
S/O sol.
0.5 h 1h 2h 4h
b
Time (h) Fig. 2. In vivo fluorescence intensity of Cy5-ovalbumin (OVA) in murine nasal cavities. (a) Relative fluorescence intensity at 0.5, 1.0, 2.0, and 4.0 h after intranasal administration of the indicated formulation with Cy5-OVA. (b) Comparisons of the amounts of Cy5-OVA adhering to the nasal cavity at 0.5 h after vaccination and at 2.0 h after vaccination. Data are expressed as the mean ± the standard deviation (n = 3).
3.3. Intranasal immunization and antibody evaluation To test whether S/O nanodispersions could strengthen systemic and mucosal immune responses, mice were vaccinated intranasally once per week for 3 weeks, then levels of OVA-specific antibody were measured via ELISA. Antibody levels in serum and nasal mucosa represent the respective development of systemic and mucosal immunity. With regard to systemic immunity, after the administration of S/O nanodispersions more IgG, IgG1, IgG2a, and IgA production was induced than in mice administered free OVA in PBS (respective increases of 2.1, 4.4, 7.0, and 2.6, all p < 0.05). With regard to mucosal immunity, there was significantly more IgA in the nasal mucosal washes from mice treated with S/O nanodispersions than in the nasal mucosal washes from mice treated with free OVA in PBS (Fig. 4). 3.4. Histopathology To assess whether S/O nanodispersions could cause structural changes in the mucosal membrane, nasal toxicity was evaluated via H& E staining. Large areas of the nasal epithelium and cilia were damaged in the noses of mice treated with IPA, suggesting high toxicity of IPA to the nasal mucosa (Fig. 5). Conversely, in the group of mice administered S/O nanodispersions the nasal cilia appeared bushy and regular, and the epithelium was intact. No removal of mucosa or cells of the epithelium was observed, which was similar to the group of mice administered PBS alone. 4. Discussion Intranasal vaccination has been investigated as an alternative to subcutaneous, intradermal, and intramuscular vaccination. Compared with subcutaneous injection, as well as inducing a systemic immune 4
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Fig. 3. Serum antibody responses in mice after intranasal vaccination. Ovalbumin (OVA) in solid-in-oil nanodispersions (S/O sol.) and phosphate-buffered saline solution (PBS sol.) were administered once per week for 3 weeks, then serum (a) OVA-specific total IgG, (b) IgG1, (c) IgG2a, and (d) IgA levels in mice were measured via ELISA. Data are expressed as the mean ± the standard deviation (n = 5 or 6). *p < 0.05. **p < 0.01.
compared with the PBS preparation. This may be attributable to the high viscosity of S/O nanodispersions. The antibody levels detected after intranasal administration in the current study suggest the potential of S/O nanodispersions in this context. To test the hypothesis that immune responses were associated with prolongation of the amount of time that antigen remained in the nasal cavity, and the adjuvanting capacity of S/O nanodispersions, antibody levels were evaluated. After intranasal administration of S/O nanodispersions in mice, total IgG, IgG1 IgG2a, and IgA levels in the serum were increased significantly compared with mice administered OVA in PBS (Fig. 3), indicating that systemic immune responses were improved. Levels of IgA in mucosal washes were also investigated, as an indicator of mucosal defense. IgA can reportedly neutralize and eliminate pathogens in the nasal mucosa, thus preventing them from reaching the mucosal epithelial barrier (Lycke, 2012). In the current study IgA levels in nasal mucosal washes after administration of S/O nanodispersions were significantly higher than those after immunization with OVA in PBS (Fig. 4). The increases in antibody levels in sera and nasal washes may have been due to the successful delivery of OVA via S/O nanodispersions. Notably, squalane oil is a principal component of the Syntex Adjuvant Formulation and DETOX (Fox, 2009). Therefore, S/O nanodispersions utilizing squalane may enhance immune responses via adjuvantation. One of the reasons why there are currently few reports describing intranasal vaccination using oil-based systems may pertain to concerns relating to potentially adverse effects on the nasal mucosa. Images of sections of nasal mucosa tissue indicated that S/O nanodispersions had no effect on the integrity of nasal epithelium and cilia (Fig. 5). The morphology of nasal mucosa after intranasal administration of S/O nanodispersions were similar with that after treatments of chitosan nanoparticle and O/W microemulsion reported previous (Khunt et al., 2017; Liu et al., 2015). Conversely, sections of nasal mucosa tissue treated with IPA exhibited damaged nasal epithelium and cilia. Therefore, S/O nanodispersions may be safe for intranasal vaccination and appropriate for use as antigenic carriers in this context.
Fig. 4. Mucosal IgA titers in mice after intranasal vaccination. Ovalbumin (OVA) in solid-in-oil nanodispersions (S/O sol.) and phosphate-buffered saline solution (PBS sol.) were administered once per week for 3 weeks, then OVAspecific IgA levels in mucosal washes were measured via ELISA. Data are expressed as the mean ± the standard deviation (n = 5 or 6). *p < 0.05.
the mucociliary clearance of antigen, was assessed as a potentially important component of its immunogenicity. Greater mucoadhesive ability could extend the amount of time antigen remains in the nasal cavity, thus facilitating stronger immune responses. Strong fluorescent signals in IVIS images indicating nasal absorption in mice suggested that the S/O nanodispersions investigated prolonged the amount of time that OVA remained in the nasal cavity (Fig. 2a). Moreover, section images of nasal cavities observed via confocal laser scanning microscopy were concordant with the IVIS images indicating that more Cy5OVA remained attached on the nasal mucosa when it was administered in the form of S/O nanodispersions (Fig. S4). The cumulative amounts of Cy5–OVA in the nasal cavity were quantified, and were concordant with these imaging results (Fig. 2b). Collectively the results of the quantitative and qualitative analysis of nasal absorption experiments indicated that S/O nanodispersions prolonged the amount of time that intranasally administered Cy5-OVA remained in the nasal cavity 5
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Fig. 5. Morphology of nasal mucosa in mice after intranasal administration of ovalbumin in solid-in-oil nanodispersions (S/O sol.), phosphate-buffered saline solution (PBS sol.), or isopropanol solution (IPA sol.). The images were captured using a fluorescence microscope with × 4, ×20, and × 40 objective lenses. Cilia are indicated by arrows.
To the best of our knowledge this is the first report describing intranasal vaccination using oil-based nanotechnology. Notably however, the mechanisms involved in the effects of intranasal immunization via S/O nanodispersions remain unclear. The main mechanism may be similar to that of water-based particle preparations in that antigens in the vaccine are transported to the immune system by specialized microfold cells in nasal-associated lymphoid tissues (Marasini, 2017; Shakya et al., 2016; Zaman et al., 2013). S/O nanodispersions maybe stable after contacting with mucin on the surface of nasal mucosa, which has an amphiphilic structure, and first form solid-in-oil-in-water emulsions (Fig. S5) and then be taken up by microfold cells. To further improve the immunogenicity of S/O nanodispersions, the incorporation of other adjuvants such as CpG oligoribodeoxynucleotides or resiquimod R848 will be investigated in the future (Kitaoka et al., 2017; Wakabayashi et al., 2018).
Funding This work was supported by a Grant-in-Aid for Scientific Research (S) 16H06369 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and also by Research Fellowships of the JSPS for Young Scientists (Q. Kong). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.118777. References Aucouturier, J., Dupuis, L., Ganne, V., 2001. Adjuvants designed for veterinary and human vaccines. Vaccine 19, 2666–2672. https://doi.org/10.1016/S0264-410X(00) 00498-9. Battaglia, L., Panciani, P.P., Muntoni, E., Capucchio, M.T., Biasibetti, E., De Bonis, P., Mioletti, S., Fontanella, M., Swaminathan, S., 2018. Lipid nanoparticles for intranasal administration: application to nose-to-brain delivery. Expert Opin. Drug Deliv. 15, 369–378. https://doi.org/10.1080/17425247.2018.1429401. Bonferoni, M., Rossi, S., Sandri, G., Ferrari, F., Gavini, E., Rassu, G., Giunchedi, P., 2019. Nanoemulsions for “nose-to-brain” drug delivery. Pharmaceutics 11, 84. https://doi. org/10.3390/pharmaceutics11020084. Fox, C.B., 2009. Squalene emulsions for parenteral vaccine and drug delivery. Molecules 14, 3286–3312. https://doi.org/10.3390/molecules14093286. Fukuyama, Y., Yuki, Y., Katakai, Y., Harada, N., Takahashi, H., Takeda, S., Mejima, M., Joo, S., Kurokawa, S., Sawada, S., Shibata, H., Park, E.J., Fujihashi, K., Briles, D.E., Yasutomi, Y., Tsukada, H., Akiyoshi, K., Kiyono, H., 2015. Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques. Mucosal Immunol. 8, 1144–1153. https://doi.org/10.1038/mi.2015.5. Ghaffar, K.A., Marasini, N., Giddam, A.K., Batzloff, M.R., Good, M.F., Skwarczynski, M., Toth, I., 2016. Liposome-based intranasal delivery of lipopeptide vaccine candidates against group A streptococcus. Acta Biomater. 41, 161–168. https://doi.org/10. 1016/j.actbio.2016.04.012. Hall, S.I., Krietz, G.A., Ulrich, R.G., Cisney, E.D., Fernandez, S., 2012. Examining the role of nasopharyngeal-associated lymphoreticular tissue (NALT) in mouse responses to vaccines. J. Vis. Exp. 1–7. https://doi.org/10.3791/3960. Jabbal-Gill, I., Watts, P., Smith, A., 2012. Chitosan-based delivery systems for mucosal vaccines. Expert Opin. Drug Deliv. 9, 1051–1067. https://doi.org/10.1517/ 17425247.2012.697455. Kantipakala, R., Bonam, S.R., Vemireddy, S., Miryala, S., Halmuthur, M., S.K., 2019. Squalane-based emulsion vaccine delivery system: composition with murabutide activate Th1 response. Pharm. Dev. Technol. 24, 269–275. https://doi.org/10.1080/ 10837450.2018.1469150. Khunt, D., Shah, B., Misra, M., 2017. Role of butter oil in brain targeted delivery of Quetiapine fumarate microemulsion via intranasal route. J. Drug Deliv. Sci. Technol.
5. Conclusion Oil-based formulations based on S/O technology exhibit potential as intranasal vaccine carriers. In the present study, in in vivo nasal absorption experiments S/O formulations were associated with comparatively longer retention of OVA in the nasal cavity, and in vivo immunization induced strong mucosal and systemic immune responses. Intranasal vaccination using S/O nanodispersions may constitute a relatively simpler method of effective en masse immunization against infectious pathogens in the future. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Acknowledgments We thank Professors Y. Katayama and T. Mori for their assistance with the animal experiments. We thank Dr Owen Proudfoot from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The authors declare no conflicts of interest. 6
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Solid-in-oil nanodispersions for intranasal vaccination: Enhancement of mucosal and systemic immune responses
T
Qingliang Konga, Momoko Kitaokaa, Yoshiro Taharaa, Rie Wakabayashia,b, Noriho Kamiyaa,b,c, ⁎ Masahiro Gotoa,b,c, a
Department of Applied Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan Advanced Transdermal Drug Delivery Center, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan c Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Immunization Intranasal vaccination Solid-in-oil nanodispersion Vaccine
En masse vaccination is a promising strategy for combatting infectious diseases. Intranasal vaccination is a viable route of mass vaccination, and it could be performed easily via needle-free administration. However, it is not widely used because it tends not to evoke sufficient immunity. The aim of the present study was to improve the performance of intranasal vaccination by extending the amount of time that administered antigens remain in the nasal cavity, and enhancing immune responses via a nanocarrier-based adjuvant. A simple and safe solid-in-oil (S/O) system was investigated as a nanocarrier in intranasal vaccination. S/O nanodispersions are oil-based dispersions of antigens coated with surfactants. Because of the mucoadhesive capacities of surfactant and oil they have high potential to extend the amount of time that administered antigens remain in the nasal cavity, and can induce strong immune responses due to a nanocarrier-based adjuvant effect. In nasal absorption experiments antigens administered intranasally via S/O nanodispersions remained in the nasal cavity longer and induced strong mucosal and systemic immune responses. Histopathology analysis indicated that S/O nanodispersions did not modify the nasal epithelium or cilia, suggesting non-toxicity of the carrier. These results indicate the potential of intranasal vaccination using S/O nanodispersions for future vaccination.
1. Introduction Frequent outbreaks and rapid spread of airborne infectious diseases such as influenza, tuberculosis, and measles are major public health problems worldwide. Vaccination is the most effective method for protecting against these infectious diseases. The common methods of vaccination via subcutaneous, intradermal, and intramuscular injection have several drawbacks however, such as low patient compliance, need for administration by a medical professional and needle-stick injuries (Narwaney et al., 2017; Trinh et al., 2017). Moreover, mass vaccination via injection is problematic after an infectious diseases has already broken out and spread worldwide, particularly in developing countries (Levine, 2003). In addition, injection can induce protective systemic antibodies but does not induce substantial mucosal antibodies, which contribute to the prevention of infectious diseases (Lycke, 2012). To overcome these issues, more suitable and effective vaccination
strategies are urgently needed. Intranasal vaccination has recently emerged as an attractive alternative to subcutaneous, intradermal, and intramuscular vaccination. Because intranasal vaccination is needle-free and non-invasive, it could be used for large-scale immunization without an inherent need for a trained medical professional. The nasal mucosa is easily accessible and highly vascularized, and has abundant immune cells with the potential to induce effective immune responses. In addition, nasal administration is reportedly able to induce both systemic and mucosal immune responses (Lycke, 2012). Since most infections start from mucosal surfaces, mucosal immunity is important as a first-line of pathogen defense. The pathogen-specific antibodies are stimulated by mucosal vaccination and secreted into the mucus to neutralize the pathogen. The main mechanism of the induction of immunity via intranasal vaccination is that antigens in the vaccine are transported via microfold cells in the nasal epithelium, then resident dendritic cells capture the antigens,
Abbreviations: ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein-4-isothiocyanate; H&E, hematoxylin and eosin; Ig, immunoglobulin; IPA, isopropanol; IVIS, in vivo imaging system; L-195, sucrose laurate; OD, optical density; OVA, ovalbumin; O/W, oil-in-water; PBS, phosphate-buffered saline; S/O, solid-in-oil; TEM, transmission electron microscopy; W/O, water-in-oil; W/O/W, water-in-oil-in-water ⁎ Corresponding author at: Department of Applied Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan. E-mail address: [email protected] (M. Goto). https://doi.org/10.1016/j.ijpharm.2019.118777 Received 8 June 2019; Received in revised form 29 September 2019; Accepted 7 October 2019 Available online 31 October 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.
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(Osaka, Japan). Perilla oil was purchased from Kaneda (Tokyo, Japan). A surfactant sucrose laurate (L-195) was kindly provided by MitsubishiKagaku Foods (Tokyo, Japan). Horseradish peroxidase-labeled rabbit anti-mouse Ig (immunoglobulin) G, IgG1, and IgG2a were obtained from Rockland Immunochemicals (Gilbertsville, PA, USA). All other reagents used were of analytical grade. Six-week-old female BALB/c mice were purchased from Kyudo, Co. (Saga, Japan) a week prior to experimentation, and maintained under standard conditions. All mouse experiments were approved by the Ethics Committee for Animal Experiments of Kyushu University, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Science Council of Japan).
process them, and induce immune responses (Marasini, 2017; Shakya et al., 2016; Zaman et al., 2013). Despite these advantages, only a few commercially available intranasal vaccines, such as Flumist®, Fluenz®, and Nasovac®, have been developed (Lycke, 2012). The main challenges include degradation of free antigen in the vaccine by enzymes in the nasal cavity, the short time that antigens remain in the nasal cavity due to rapid nasal mucociliary clearance, and weak immune responses resulting from inefficient antigen transportation to the immune system (Marasini, 2017). Therefore, the development of an effective vaccine formulation that can maintain antigen stability, extend the amount time that antigen stays in the nasal cavity, and stimulate immune responses as an adjuvant is required. To date, nanogel (Fukuyama et al., 2015; Nochi et al., 2010), liposomes (Ghaffar et al., 2016; Tada et al., 2015), emulsions (Makidon et al., 2012; Wong et al., 2014), lipid (Battaglia et al., 2018), and polymeric nanoparticles (Lebre et al., 2016; Pawar and Jaganathan, 2016) have been developed as effective formulations for intranasal vaccination. Of these, emulsions have attracted much attention due to the ease of manufacturing them and scaling up production, and the adjuvant effect caused by constituent surfactants and oils (Fox, 2009; Wong et al., 2014). Notably however, oil-in-water (O/W) emulsions—which are the most common emulsions used in intranasal vaccines—have several drawbacks including the short amount of time that antigen remains in the nasal cavity due to the low viscosity of waterbased emulsions, and low loading efficiency of antigens because they are simply mixed with oil-droplets and not encapsulated inside oil. In some cases, bursting or incomplete release causes only part of the antigen to be transported to the immune system (Aucouturier et al., 2001; Mahajan and Rasal, 2013). In an effort to overcome these issues, intranasal vaccination involving a solid-in-oil (S/O) nanodispersion technique was investigated in the present study. S/O nanodispersions are oil-based dispersions containing nano-sized particles consisting of antigen coated by hydrophobic surfactant molecules (Kitaoka et al., 2016). In contrast to waterbased emulsions such as O/W and water-in-O/W (W/O/W) emulsions which have traditionally been used in intranasal vaccination, oil-based S/O nanodispersions exhibit high stability for more than 3 months due to reduced opportunity for Ostwald ripening (Kitaoka et al., 2016), and high antigen encapsulation efficiency of up to 99.9% (Wakabayashi et al., 2018). Moreover, as well as extending the amount of time that antigen remains in the nasal cavity such that it is transported to the immune system, the oil-based formulation of S/O nanodispersions can reportedly act as an adjuvant and strengthen immune responses (Fox, 2009). To the best of our knowledge few oil-based formulations have been developed for intranasal vaccination (Bonferoni et al., 2019), and only the delivery of insulin via the nasal route using water-in-oil (W/O) microemulsions has been reported (Sintov et al., 2010). In the current study the potential of intranasal vaccination using S/ O nanodispersions loaded with the model antigen ovalbumin (OVA) was investigated. S/O nanodispersions were prepared using different oils, and formulation optimization was attempted. Nasal absorption and the amount of time that OVA remained in the nasal cavity after administration in an S/O nanodispersion were assessed. S/O nanodispersions were administered to mice intranasally for the evaluation of OVA-specific immune responses. Lastly, the health of the nasal mucosa was assessed after intranasal administration of S/O nanodispersions.
2.2. Preparation of S/O nanodispersions in different oils S/O nanodispersions were prepared as previously described (Kitaoka et al., 2016). A W/O emulsion was prepared from an aqueous OVA solution (0.5 mg/mL) and a cyclohexane solution of surfactant L195 (12.5 mg/mL) using a PT2500E polytron homogenizer (Kinematica AG, Luzern, Switzerland) at 26,000 rpm for 2 min. The W/O emulsion was flash-frozen in liquid nitrogen for 20 min then lyophilized for 24 h with an FDU-1200 lyophilizer (Eyela, Tokyo, Japan). Lastly, the resulting viscous surfactant-OVA complex was dispersed in squalane, linseed, perilla, or soybean oil to yield the S/O nanodispersions (Fig. S1). The size distribution of the S/O nanodispersions was analyzed with a Zetasizer Nano ZS light scattering instrument (Malvern, Worcestershire, UK). The viscosity of the S/O nanodispersions was measured with a microviscometer (Lovis 2000 M/ME, Anton Paar, Austria). Morphological analysis was performed by transmission electron microscopy (TEM) using a JEM-2010 (JEOL, Tokyo, Japan). Briefly, the specimen was stained with 2% uranyl acetate, dried in vacuo, and imaged at an accelerating voltage of 120 kV. 2.3. Drug release test Drug release tests were performed using custom-fabricated Franztype diffusion cells with an effective diffusion area of 0.785 cm2 and a receptor volume of 5 mL. A polycarbonate film (Whatman Nuclepore Track-Etch Membrane, 0.1 µm; GE Healthcare) was set on a cell, and the receptor compartment was filled with PBS pH 6.4 which is within the normal murine nasal cavity pH range (Shah et al., 2017). S/O nanodispersions carrying 1 mg/mL of FITC-labeled OVA dispersed in different oils (0.25 mL) were added to the donor phase of Franz-type diffusion cell and the cell was incubated for 6 h at 34 °C, which is within the normal murine nasal cavity temperature range. Samples were extracted from the receptor compartment at 1, 3, and 6 h, and replaced with the same volume of fresh media. FITC-labeled OVA concentrations were measured with a fluorescence spectrometer LS-55 (PerkinElmer, Waltham, MA, USA). 2.4. In vivo fluorescence imaging Mice under anesthesia were intranasally administered 10 μL of PBS solution or S/O nanodispersions containing Cy5-OVA. After 0.5 h, 1.0 h, 2.0 h, and 4.0 h mice were scanned using an in vivo imaging system (IVIS-Lumina II-FS-T3 Xenogen, USA) at an excitation wavelength of 640 nm. Mice administered PBS solution alone were used as controls.
2. Materials and methods 2.5. In vivo nasal absorption 2.1. Materials To investigate the amount of Cy5-OVA adhered to nasal tissue, the nasal cavities of mice were isolated at 0.5 and 2.0 h after intranasal administration of Cy5-OVA PBS solution or S/O nanodispersions. The nasal surface was then washed with an extraction solution consisting of a mixture of PBS, methanol, and acetonitrile (2:1:1 v/v/v) to remove Cy5-OVA from the nasal surface. The nasal tissue was then cut into
OVA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein-4-isothiocyanate (FITC) was purchased from Dojindo (Kumamoto, Japan), and Cy5 mono-reactive dye was purchased from GE Healthcare (UK). Cyclohexane, linseed oil, soybean oil, and squalane oil were obtained from Fujifilm Wako Pure Chemical Corporation 2
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pieces and Cy5-OVA was extracted with extraction solution for 24 h. The cumulative amounts of Cy5-OVA were calculated based on fluorescence intensities measured via the LS55 fluorescence spectrometer.
Table 1 Characterization of solid-in-oil nanodispersions in different oils. S/O in different oils
Diameter (nm)
PDI
Viscosity (mPa s, 25 °C)
Viscosity (mPa s, 34 °C)
Squalane Linseed Soybean Perilla
148.4 ± 6 327 ± 15 958 ± 20 1234 ± 111
0.170–0.241 0.125–0.298 0.044–0.169 0.280–0.348
31.36 38.76 55.58 46.99
21.17 28.30 38.66 32.79
2.6. Murine intranasal immunization Mice were randomly divided into two groups and immunized with OVA-PBS or S/O nanodispersions (10 μL each, 2 mg/mL) once per week for 3 weeks. Mice were held upright immediately after vaccination, to ensure maximal dosing and prevent swallowing. Blood samples were collected from a tail vein 7 days after the third immunization. All mice were then killed via neck dislocation, and nasal mucosal washes were collected through the trachea toward the nasal cavity with 0.5 mL of PBS (Puchta et al., 2014). 2.7. Measurement of serum and mucosal wash antibody levels Serum from immunized mice was assayed via the enzyme-linked immunosorbent assay (ELISA) to determine levels of OVA-specific antibodies (total IgG, IgG1, IgG2a, and IgA) as previously described (Tahara et al., 2010). The antibody titer was defined as the dilution factor at which the optical density of the sample was equal to that of a non-immunized sample. OVA-specific IgA levels in mucosal washes were assayed via ELISA using the Mouse IgA Ready-SET-Go kit (eBioscience, San Diego, CA, USA). Optical density (OD) was read at wavelengths of 450 nm and 570 nm on an iMark microplate reader (Bio-Rad, Berkeley, CA, USA).
Fig. 1. Cumulative release (%) of ovalbumin (OVA) from solid-in-oil nanodispersions into aqueous media. Each point represents the mean ± standard deviation of four replicates. The data reflect percentages of the cumulative amounts of OVA in the receptor chambers.
2.8. Histopathology 34 °C. However, the viscosity of S/O nanodispersions was increased more than 30 times compared with PBS solution. S/O nanodispersions in perilla and soybean oil exhibited the highest viscosity, but S/O in squalane oil exhibited comparatively low viscosity (Table 1). The OVA release efficiency of S/O nanodispersions was evaluated using a Franz diffusion cell instrument. For antigen to be effective it must be released from S/O nanodispersions. Complete release of the OVA was observed within 6 h for the S/O dispersed in squalane oil (Fig. 1). Conversely, less than 15% of the OVA was released from the S/ O dispersed in other oils within 6 h.
The nasal toxicity of S/O nanodispersions was evaluated via hematoxylin and eosin (H&E) staining. Mice received S/O nanodispersions (10 μL) via intranasal instillation. OVA PBS solution was used as a positive control and OVA isopropanol (IPA) solution was used as a negative control (Khunt et al., 2017). At 1 h after administration the mice were killed via an overdose of anesthetic, and the cranium was detached from the vertebrae. The cranium was fixed with 4% paraformaldehyde for 24 h. The nasal cavities of the mice were carefully excised, decalcified with 10% formic acid for 12 h, embedded in optimal cutting temperature compound, then stained with H&E and observed using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) (Hall et al., 2012).
3.2. Nasal absorption of antigen To examine the amount of time that OVA in S/O nanodispersions remained in the nasal cavity, in vivo nasal absorption of Cy5-OVA in mice was evaluated using an IVIS. Mice were intranasally administered the Cy5-OVA in a PBS solution or in S/O nanodispersions, and views of living animals were recorded at predetermined time-points (Fig. 2a). When Cy5-OVA in PBS was administered there was rapid clearance of Cy5-OVA at the site of administration, and almost no fluorescent signal was detected in the nose even at 1 h. However, strong fluorescence was detected in the noses of mice treated with Cy5-OVA S/O nanodispersion, and some signal was detected even at 4 h. To assess differences in amounts of Cy5-OVA adhered to the nasal mucosa, sections of nasal cavities were imaged with a confocal laser scanning microscope. The fluorescent signal in mice treated with S/O nanodispersions was stronger than that of mice treated with PBS solution at 0.5 h, 2.0 h, and 4.0 h (Fig. S4). The amounts of Cy5-OVA remaining in the nasal cavity after vaccination were quantified. At 0.5 h after vaccination S/O nanodispersions exhibited the mucosal adhesion of more than twice the amount of OVA than the PBS solution, and at 2.0 h it was more than four times the amount (Fig. 2b). Collectively the results indicated that S/O nanodispersions prolonged the intranasal retention of OVA in the nasal cavity.
2.9. Statistical analysis The data were expressed as means ± the standard deviation. The ttest was used in statistical analysis, which was performed using GraphPad Prism software. 3. Results 3.1. Characterization of the S/O nanodispersions in the different oils The physicochemical characterization of S/O nanodispersions can be defined by various parameters such as physical appearance, particle size, viscosity, and release efficiency. The physical appearance of S/O nanodispersions containing OVA was transparent and homogeneous in the different oils (Fig. S2). Morphological investigation using TEM suggested that spherical structure of the surfactant-peptide complex was formed (Fig. S3). The size distribution of S/O nanodispersions as measured via dynamic light scattering suggested that the respective mean diameters were 148, 327, and more than 1000 nm in squalane oil, linseed oil, and perilla and soybean oil S/O nanodispersions (Table 1). The viscosity of S/O nanodispersions in different oils was measured using a microviscometer at 25 °C and 34 °C. The OVA in PBS solution exhibited low viscosities of 0.907 mPa s at 25 °C and 0.753 mPa s at 3
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a
Control
PBS sol.
response it also increases local mucosal immune responses, which contribute to blocking initial infection and prevent further infection. Due to the poor immunogenicity of free antigens in intranasal vaccines however, more effective vaccine formulations are required. Nanoparticle systems are commonly used to improve the immunogenicity of antigens because they can facilitate the efficient transport of those antigens to the immune system (Marasini, 2017). Among nanoparticle systems, emulsions—particularly O/W nanoemulsions—have attracted much attention due to the ease of manufacturing and scaling up, and the adjuvanting effects of surfactants and oils (Fox, 2009; Wong et al., 2014). S/O nanodispersions have been developed from W/O emulsions as an oil-based dispersion of nano-sized particles. They are prepared by removing water from a W/O emulsion via lyophilization, and redispersing the surfactant/antigen complex in another oil vehicle. Compared with conventional O/W emulsions, S/O nanodispersions exhibit higher stability and higher antigen encapsulation efficiency. We have previously assessed immune responses to transcutaneous immunization with S/O nanodispersions loaded with OVA (Kitaoka et al., 2014), and more recently we developed pollinosis and cancer immunotherapies using S/O nanodispersions (Kong et al., 2017; Wakabayashi et al., 2018). In our previous studies S/O nanodispersions with an entirely oil base had high viscosity and strong adjuvanting effects compared with free antigen in PBS. Moreover, high viscosity and adjuvant effects are considered essential for intranasal vaccines, which require high mucoadhesive ability in order to ensure that the amount of time antigen remains in the nasal cavity is sufficient to facilitate transport to the immune system, and adjuvanting to strengthen immune responses. Therefore, we hypothesized that S/O nanodispersions may be useful for intranasal vaccination. One crucial aspect of the formulation of S/O nanodispersions for intranasal vaccination is the choice of oil, which determines the size, mucoadhesive capacity, and adjuvanting effect of the formulation. Highly purified non-mineral oils are reportedly well tolerated because they are rapidly metabolized and eliminated (Aucouturier et al., 2001). Squalane oil was deemed to be the optimal oil for the formulation in the present study due to nano-sized particle formation, comparable viscosity, and complete release of antigen. S/O dispersions using squalane oil exhibited a nano-level mean diameter, whereas those using perilla and soybean oil exhibited micro-level mean diameters (Table 1). Nanosized vaccines are preferable for intranasal vaccination because they are associated with more efficient transport of antigen to the immune system (Marasini, 2017). The viscosities of the formulations investigated were evaluated as a potential indicator of mucoadhesive capacity. The viscosities of the S/O nanodispersions were at least 30 times greater than that of the PBS solution, which exhibited viscosity of 0.907 mPa s at 25 °C and 0.753 mPa s at 34 °C (Table 1). Different from water-based chitosan nanoparticles and cationic liposomes, their positive charge binding to the negatively charged mucus (Jabbal-Gill et al., 2012; Tada et al., 2015), the greater viscosity of S/O nanodispersions could promote adhesion to the nasal mucosal surface. Complete release of OVA from S/O nanodispersions in squalane oil occurred within 6 h, while the amount of OVA dispersed in other oils released within 6 h was less than 15% (Fig. 1). Considering the possibility of incomplete antigen release and the rapid clearance of antigen in the intranasal cavity (Riese et al., 2014), complete release within 6 h was considered suitable for an intranasal vaccination. There are several reports of the formulation of stable emulsions based on squalene oil in clinic studies, and in its hydrogenated form it has been preferred due to its high chemical stability, resistance to oxidation, and metabolizing properties (Fox, 2009; Kantipakala et al., 2019). Mechanisms of transdermal antigen delivery via S/O nanodispersions have also been investigated in a previous study (Kitaoka et al., 2014). The normal nasal mucociliary clearance time is only approximately 20 min in humans (Soane et al., 1999). Therefore each intranasal vaccine formulation’s capacity for mucoadhesion, which could slow down
S/O sol.
0.5 h 1h 2h 4h
b
Time (h) Fig. 2. In vivo fluorescence intensity of Cy5-ovalbumin (OVA) in murine nasal cavities. (a) Relative fluorescence intensity at 0.5, 1.0, 2.0, and 4.0 h after intranasal administration of the indicated formulation with Cy5-OVA. (b) Comparisons of the amounts of Cy5-OVA adhering to the nasal cavity at 0.5 h after vaccination and at 2.0 h after vaccination. Data are expressed as the mean ± the standard deviation (n = 3).
3.3. Intranasal immunization and antibody evaluation To test whether S/O nanodispersions could strengthen systemic and mucosal immune responses, mice were vaccinated intranasally once per week for 3 weeks, then levels of OVA-specific antibody were measured via ELISA. Antibody levels in serum and nasal mucosa represent the respective development of systemic and mucosal immunity. With regard to systemic immunity, after the administration of S/O nanodispersions more IgG, IgG1, IgG2a, and IgA production was induced than in mice administered free OVA in PBS (respective increases of 2.1, 4.4, 7.0, and 2.6, all p < 0.05). With regard to mucosal immunity, there was significantly more IgA in the nasal mucosal washes from mice treated with S/O nanodispersions than in the nasal mucosal washes from mice treated with free OVA in PBS (Fig. 4). 3.4. Histopathology To assess whether S/O nanodispersions could cause structural changes in the mucosal membrane, nasal toxicity was evaluated via H& E staining. Large areas of the nasal epithelium and cilia were damaged in the noses of mice treated with IPA, suggesting high toxicity of IPA to the nasal mucosa (Fig. 5). Conversely, in the group of mice administered S/O nanodispersions the nasal cilia appeared bushy and regular, and the epithelium was intact. No removal of mucosa or cells of the epithelium was observed, which was similar to the group of mice administered PBS alone. 4. Discussion Intranasal vaccination has been investigated as an alternative to subcutaneous, intradermal, and intramuscular vaccination. Compared with subcutaneous injection, as well as inducing a systemic immune 4
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Fig. 3. Serum antibody responses in mice after intranasal vaccination. Ovalbumin (OVA) in solid-in-oil nanodispersions (S/O sol.) and phosphate-buffered saline solution (PBS sol.) were administered once per week for 3 weeks, then serum (a) OVA-specific total IgG, (b) IgG1, (c) IgG2a, and (d) IgA levels in mice were measured via ELISA. Data are expressed as the mean ± the standard deviation (n = 5 or 6). *p < 0.05. **p < 0.01.
compared with the PBS preparation. This may be attributable to the high viscosity of S/O nanodispersions. The antibody levels detected after intranasal administration in the current study suggest the potential of S/O nanodispersions in this context. To test the hypothesis that immune responses were associated with prolongation of the amount of time that antigen remained in the nasal cavity, and the adjuvanting capacity of S/O nanodispersions, antibody levels were evaluated. After intranasal administration of S/O nanodispersions in mice, total IgG, IgG1 IgG2a, and IgA levels in the serum were increased significantly compared with mice administered OVA in PBS (Fig. 3), indicating that systemic immune responses were improved. Levels of IgA in mucosal washes were also investigated, as an indicator of mucosal defense. IgA can reportedly neutralize and eliminate pathogens in the nasal mucosa, thus preventing them from reaching the mucosal epithelial barrier (Lycke, 2012). In the current study IgA levels in nasal mucosal washes after administration of S/O nanodispersions were significantly higher than those after immunization with OVA in PBS (Fig. 4). The increases in antibody levels in sera and nasal washes may have been due to the successful delivery of OVA via S/O nanodispersions. Notably, squalane oil is a principal component of the Syntex Adjuvant Formulation and DETOX (Fox, 2009). Therefore, S/O nanodispersions utilizing squalane may enhance immune responses via adjuvantation. One of the reasons why there are currently few reports describing intranasal vaccination using oil-based systems may pertain to concerns relating to potentially adverse effects on the nasal mucosa. Images of sections of nasal mucosa tissue indicated that S/O nanodispersions had no effect on the integrity of nasal epithelium and cilia (Fig. 5). The morphology of nasal mucosa after intranasal administration of S/O nanodispersions were similar with that after treatments of chitosan nanoparticle and O/W microemulsion reported previous (Khunt et al., 2017; Liu et al., 2015). Conversely, sections of nasal mucosa tissue treated with IPA exhibited damaged nasal epithelium and cilia. Therefore, S/O nanodispersions may be safe for intranasal vaccination and appropriate for use as antigenic carriers in this context.
Fig. 4. Mucosal IgA titers in mice after intranasal vaccination. Ovalbumin (OVA) in solid-in-oil nanodispersions (S/O sol.) and phosphate-buffered saline solution (PBS sol.) were administered once per week for 3 weeks, then OVAspecific IgA levels in mucosal washes were measured via ELISA. Data are expressed as the mean ± the standard deviation (n = 5 or 6). *p < 0.05.
the mucociliary clearance of antigen, was assessed as a potentially important component of its immunogenicity. Greater mucoadhesive ability could extend the amount of time antigen remains in the nasal cavity, thus facilitating stronger immune responses. Strong fluorescent signals in IVIS images indicating nasal absorption in mice suggested that the S/O nanodispersions investigated prolonged the amount of time that OVA remained in the nasal cavity (Fig. 2a). Moreover, section images of nasal cavities observed via confocal laser scanning microscopy were concordant with the IVIS images indicating that more Cy5OVA remained attached on the nasal mucosa when it was administered in the form of S/O nanodispersions (Fig. S4). The cumulative amounts of Cy5–OVA in the nasal cavity were quantified, and were concordant with these imaging results (Fig. 2b). Collectively the results of the quantitative and qualitative analysis of nasal absorption experiments indicated that S/O nanodispersions prolonged the amount of time that intranasally administered Cy5-OVA remained in the nasal cavity 5
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Fig. 5. Morphology of nasal mucosa in mice after intranasal administration of ovalbumin in solid-in-oil nanodispersions (S/O sol.), phosphate-buffered saline solution (PBS sol.), or isopropanol solution (IPA sol.). The images were captured using a fluorescence microscope with × 4, ×20, and × 40 objective lenses. Cilia are indicated by arrows.
To the best of our knowledge this is the first report describing intranasal vaccination using oil-based nanotechnology. Notably however, the mechanisms involved in the effects of intranasal immunization via S/O nanodispersions remain unclear. The main mechanism may be similar to that of water-based particle preparations in that antigens in the vaccine are transported to the immune system by specialized microfold cells in nasal-associated lymphoid tissues (Marasini, 2017; Shakya et al., 2016; Zaman et al., 2013). S/O nanodispersions maybe stable after contacting with mucin on the surface of nasal mucosa, which has an amphiphilic structure, and first form solid-in-oil-in-water emulsions (Fig. S5) and then be taken up by microfold cells. To further improve the immunogenicity of S/O nanodispersions, the incorporation of other adjuvants such as CpG oligoribodeoxynucleotides or resiquimod R848 will be investigated in the future (Kitaoka et al., 2017; Wakabayashi et al., 2018).
Funding This work was supported by a Grant-in-Aid for Scientific Research (S) 16H06369 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and also by Research Fellowships of the JSPS for Young Scientists (Q. Kong). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.118777. References Aucouturier, J., Dupuis, L., Ganne, V., 2001. Adjuvants designed for veterinary and human vaccines. Vaccine 19, 2666–2672. https://doi.org/10.1016/S0264-410X(00) 00498-9. Battaglia, L., Panciani, P.P., Muntoni, E., Capucchio, M.T., Biasibetti, E., De Bonis, P., Mioletti, S., Fontanella, M., Swaminathan, S., 2018. Lipid nanoparticles for intranasal administration: application to nose-to-brain delivery. Expert Opin. Drug Deliv. 15, 369–378. https://doi.org/10.1080/17425247.2018.1429401. Bonferoni, M., Rossi, S., Sandri, G., Ferrari, F., Gavini, E., Rassu, G., Giunchedi, P., 2019. Nanoemulsions for “nose-to-brain” drug delivery. Pharmaceutics 11, 84. https://doi. org/10.3390/pharmaceutics11020084. Fox, C.B., 2009. Squalene emulsions for parenteral vaccine and drug delivery. Molecules 14, 3286–3312. https://doi.org/10.3390/molecules14093286. Fukuyama, Y., Yuki, Y., Katakai, Y., Harada, N., Takahashi, H., Takeda, S., Mejima, M., Joo, S., Kurokawa, S., Sawada, S., Shibata, H., Park, E.J., Fujihashi, K., Briles, D.E., Yasutomi, Y., Tsukada, H., Akiyoshi, K., Kiyono, H., 2015. Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques. Mucosal Immunol. 8, 1144–1153. https://doi.org/10.1038/mi.2015.5. Ghaffar, K.A., Marasini, N., Giddam, A.K., Batzloff, M.R., Good, M.F., Skwarczynski, M., Toth, I., 2016. Liposome-based intranasal delivery of lipopeptide vaccine candidates against group A streptococcus. Acta Biomater. 41, 161–168. https://doi.org/10. 1016/j.actbio.2016.04.012. Hall, S.I., Krietz, G.A., Ulrich, R.G., Cisney, E.D., Fernandez, S., 2012. Examining the role of nasopharyngeal-associated lymphoreticular tissue (NALT) in mouse responses to vaccines. J. Vis. Exp. 1–7. https://doi.org/10.3791/3960. Jabbal-Gill, I., Watts, P., Smith, A., 2012. Chitosan-based delivery systems for mucosal vaccines. Expert Opin. Drug Deliv. 9, 1051–1067. https://doi.org/10.1517/ 17425247.2012.697455. Kantipakala, R., Bonam, S.R., Vemireddy, S., Miryala, S., Halmuthur, M., S.K., 2019. Squalane-based emulsion vaccine delivery system: composition with murabutide activate Th1 response. Pharm. Dev. Technol. 24, 269–275. https://doi.org/10.1080/ 10837450.2018.1469150. Khunt, D., Shah, B., Misra, M., 2017. Role of butter oil in brain targeted delivery of Quetiapine fumarate microemulsion via intranasal route. J. Drug Deliv. Sci. Technol.
5. Conclusion Oil-based formulations based on S/O technology exhibit potential as intranasal vaccine carriers. In the present study, in in vivo nasal absorption experiments S/O formulations were associated with comparatively longer retention of OVA in the nasal cavity, and in vivo immunization induced strong mucosal and systemic immune responses. Intranasal vaccination using S/O nanodispersions may constitute a relatively simpler method of effective en masse immunization against infectious pathogens in the future. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Acknowledgments We thank Professors Y. Katayama and T. Mori for their assistance with the animal experiments. We thank Dr Owen Proudfoot from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The authors declare no conflicts of interest. 6
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