http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.873448
RESEARCH ARTICLE
Development of Pasteurella multocida-loaded microparticles for hemorrhagic septicemia vaccine Drug Dev Ind Pharm Downloaded from informahealthcare.com by York University Libraries on 01/01/15 For personal use only.
Pataranapa Nimtrakul1,2, Ratchanee Atthi3, Nanteetip Limpeanchob4, and Waree Tiyaboonchai1,2 1
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand, 2Center of Excellence for Innovation in Chemistry, Commission on Higher Education, Bangkok, Thailand, 3Department of Livestock Development, Bureau of Veterinary Biologics, Pakchong, Nakhonratchasima, Thailand, and 4Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand Abstract
Keywords
In this study, Pasteurella multocida-loaded alginate microparticles (MPs) for subcutaneous vaccination was developed by emulsification-cross-linking technique. Formulation parameter was varied as a ratio of polymer and bacterin. Optical microscopy revealed spherical particles with uniformly distribution. A mean particle size of approximately 6 mm has been successfully constructed using simple mixer and ultrasonic probe. The zeta potential of the MPs showed negatively charge of approximately 23 mV determined by Zeta PalsÕ analyzer. The entrapment efficiency and the in vitro bacterin released profile could be controlled by varying the amount of alginate. The high entrapment efficiency up to 69% was achieved with low concentration of alginate. The MPs possessed a slow bacterin release profile, up to 30 days. In vivo safety and potency tests were proved that the alginate MPs were safe and induced protective immunity in mice. In addition, after storage for 6 months at either 4 C or room temperature, the protective immunity in mice was maintained.
Alginate, hemorrhagic septicemia, microparticles, Pasteurella multocida, vaccine
Introduction Hemorrhagic septicemia (HS) caused by infection with Pasteurella multocida (P. multocida) is a commonly fatal systemic disease of cattle and buffaloes. The disease is prevalent in South and Southeast Asia, serotype B: 2 and in Tropical Africa, serotype E: 21. An outbreak of HS, usually acute, widespread and virulently fatal, would cause economical damage and threaten the cattle export industry. To date, vaccination is the most effective means to prevent P. multocida outbreak. Alum precipitated vaccines (APVs), inactivated vaccine, is the most widely used as it is simple to manufacture. However, APVs provide short term protection, 6 months and post-vaccination shock has been reported up to 10%. In contrast, oil adjuvant vaccines provide longer protection for 1 year. However, these vaccines have disadvantages of high viscosity and post-vaccination high fever in cattle, which make it unpopular among the field users. Some attempts have been done to reduce formulation viscosity, but such vaccines suffer from high number of inactivated cells (1010– 1011)2,3. To overcome those problems, it was developed a new vaccine delivery system possesses lower viscosity, leading to lower side effects during and after injection. Microencapsulation is a unique way to deliver antigens and facilitates their uptake into lymphoid tissue. One of the most
Address for correspondence: Waree Tiyaboonchai, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand. Tel:+66-55-961873. E-mail: [email protected]
History Received 15 August 2013 Revised 27 November 2013 Accepted 2 December 2013 Published online 31 December 2013
common materials used to encapsulate antigens is alginate. It is generally regarded as safe by the United State Food and Drug Administration (FDA) and finding widespread application in pharmaceutical industry4–6. Alginate is a biodegradable, biocompatible and water soluble linear polysaccharide extracted from brown algae. It is composed of alternating block of 1–4 linked a-L-guluronic acid and b-D-mannuronic acid residue7. The parenteral administration of alginate microparticles (MPs) has been shown to provoke immune responses8–13. In the previous study, the alginate MPs were successfully developed by emulsification-cross-linking technique using calcium and zinc as a cross-linking agent14. Unfortunately, a toxicity of this formulation was observed in mice as a result from zinc. Thus, in this study, to improve the safety of MPs, we further developed antigen-loaded MPs using only calcium as a cross-linking agent. The physicochemical properties of MPs were characterized including morphology, mean particle size, zeta potential and entrapment efficiency. The profile of bacterin release was investigated as well as the stability of MPs. Moreover, the safety and efficiency of prepared vaccine was evaluated in vivo.
Materials and methods Materials Sodium alginate, medium viscosity, was purchased from Fluka (Steinheim, UK). PluronicÕ L61 was obtained from BASF (Florham Park, NJ). Olive oil was purchased from TCFF (Bangkok, Thailand). Calcium chloride was purchased from Univar (New South Wales, Australia). Sodium citrate was
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purchased from MERCK (Damstadt, Germany). A formalininactivated whole culture of P. multocida, serotype B:2, 5 was obtained from the Bureau of Veterinary Biologics, Ministry of Agriculture and Cooperatives, Thailand. Micro-BCA protein assay kit was purchased from Thermo Scientific (Rockford, IL). All other chemicals and solvents were of analytical grade.
incubating with 4% w/v sodium citrate. Finally, the amount of bacterin was determined using micro-BCA protein assay kit as described before. The percentage of bacterin released was determined by indirect method according to the equation: Bacterin release ð%Þ 1 Amount of bacterin remained in particle 100 ¼ Initial amount of bacterin
ð2Þ
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Methods Preparation of P. multocida-loaded alginate MPs
In vivo safety and potency test
Pasteurella multocida containing alginate MPs were prepared by emulsification-cross-linking method. The formalin-killed cultures were washed with de-ionized (DI) water before mixed with 2%w/v alginate solution. The alginate mixture was added to oil phase, composed of PluronicÕ L61 in olive oil, while stirred at 1500 rpm using a mixer (RZR 2021, HEIDOLPH Instruments GmbH & Co KG, Germany). The internal droplets were further reduced using ultrasonic probe. Then, cross-linking solution was added drop wise to the emulsion while stirred at 1500 rpm. The resulting antigen MPs were harvested by centrifugation at 1500 rpm for 5 min and washed three times with DI water. Then, MPs were filtrated by a 45 mm sieve. Finally, MPs were adjusted to 40 ml with DI water and kept at 4 C before used.
In vivo experiments were conducted under ethic approval from the Bureau of Veterinary Biologics, Animal Ethics Committee.
Physicochemical characterization of MPs The morphology of both P. multocida-loaded and unloaded microparticles were investigated using scanning electron microscopy (SEM). The mean particle size of the MPs was determined by an optical microscope. At least 300 particles were measured using Feret’s diameter. Cumulative percentage, frequency, undersize and normalized Z-value were calculated. To calculate the geometric mean diameter (D50), particle diameter value were transformed into logarithm value. The zeta potential of the MPs was determined by phase analysis light scattering employing a Zeta PalsÕ (Brookhaven Instruments Corporation, New York). The zeta potential was calculated from the electrophoretic mobility using the Smoluchowski approximation approach15. Determination of bacterin entrapment efficiency MPs were incubated with 4% w/v sodium citrate solution. After dissolution of the particles, samples were centrifuged at 12 000 rpm for 5 min. The resulting bacterin pellets were washed three times with DI water and re-suspended in 1 ml of DI water. Then, the amount of protein referring to bacterin was determined using the bicinchoninic acid (micro-BCA) protein assay in 96-well plates following manufacturer’s instruction. The percent bacterin entrapment efficiency was calculated according to the equation: Bacterin entrapment ð%Þ Amount of bacterin in particle 100 ¼ Initial amount of bacterin
Safety test. The safety test was conducted with acute toxicity testing in mice according to the procedure in compliance with the ASEAN standards of animal vaccines16. Ten male ICR mice, 6–8 weeks of age, were each injected intraperitoneally with 0.5 ml of the sample and were observed daily for 2 weeks. Potency test. The efficiency of MPs vaccines were evaluated by active mouse protection test17. Male ICR mice, 6–8 weeks of age, were equally divided into immunized and control group. Mice in immunized group were inoculated twice with 0.2 ml MPs vaccines by intraperitoneal (IP) route in 2 weeks interval. Mice in control group were not inoculated. One week after the second inoculation, all mice were divided into six subgroups of five mice. Each subgroup was challenged by intraperitoneal inoculation with 0.1 ml of respective dilutions of 6-h broth culture of P. multocida serotype B: 2, 5 in the range of 101 to 106 in immunized group and 104 to 109 in control group. Mice were daily observed for 1 week and lethal dose 50% (LD50) were calculated by the method of Reed & Muench18 according to the equation: Proportional distance Mortality above 50% 50 ¼ Mortality above 50% Mortality below 50%
Log of LD50 ¼ Log of dilution above 50% mortality þ Proportional distance
ð3Þ
ð4Þ
Stability test The stability of MPs in terms of physical and chemical properties was studied both 4 C and room temperature for 6 months. The physical stability of MPs was determined in terms of morphology, particles size, zeta potential and bacterin content as described before. In vivo efficiency studies were determined in terms of potency test.
Results ð1Þ
In vitro release study of bacterin The in vitro release study was performed by keeping samples in the dark at 37 0.5 C for 1 month. Five hundred microliters of MPs were placed in 0.5 ml of 0.2 M HEPES buffer, pH 7.4. At selected time interval (1, 3, 5, 7, 15 and 30 days), the amount of bacterin remained in the MPs were determined. The samples were centrifuged at 3000 rpm for 5 min to collected MPs and remove the released bacterin. Then, MPs were dissolved by
Physicochemical characterization The alginate based MPs were examined by optical microscope and SEM (Figure 1). Optical micrographs revealed that both bacterinloaded and -unloaded MPs were spherical in shape and uniformly distributed. The dark round-like structure seen in the MPs was attributed to the presence of olive oil. Similarly, SEM micrographs of both bacterin-loaded and -unloaded MPs also showed spherical particles with smooth surface. The mean particle size and zeta potential were found to be independent on ratio of polymer and bacterin. All formulations possessed similar mean particle size of approximately 6 mm and zeta potential value of approximately 23 mV (Table 1).
P. multocida-loaded alginate microparticles
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DOI: 10.3109/03639045.2013.873448
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Figure 1. Optical micrographs of formulation E (1.5:1) (A) blank MPs, (B) bacterin-loaded MPs (400 magnifications) and Scanning electron micrographs of formulation E (1.5:1) (C) blank MPs and (D) bacterin-loaded MPs (800 magnifications). Table 1. Mean particle size and zeta potential of MPs prepared with different ratio of alginate and bacterin (n ¼ 3). D50 (mm) SD Formulation A B C D E F
Zeta potential (mV) SD
Ratio of alginate:bacterin
Blank MPs
Bacterin-loaded MPs
Blank MPs
Bacterin-loaded MPs
8:1 4:1 3:1 2:1 1.5:1 1:1
6.50 0.14 6.70 0.10 5.97 0.21 5.80 0.40 5.90 0.10 6.03 0.32
6.10 0.14 6.13 0.49 5.97 0.38 5.97 0.25 5.83 0.21 5.77 0.21
20.39 1.51 22.22 0.87 22.15 0.72 21.70 0.27 23.23 1.05 21.14 0.78
24.21 1.44 23.40 0.51 22.93 0.88 22.78 0.58 23.07 1.70 22.92 1.71
Bacterin entrapment efficiency MPs were prepared by varying the ratio of alginate and bacterin, while the amount of calcium chloride kept constant. As the amount of alginate increased, formulation A (8:1) shows the lowest entrapment efficiency of 15% was observed (Figure 2). In contrast, formulation B (4:1) and C (3:1) showed entrapment efficiency of 43 and 61%, respectively. The high entrapment efficiency up to 69% was observed with formulation D (2:1), E (1.5:1) and F (1:1). Nevertheless, application of hardening, washing and filtering of MPs during production could potentially decrease the entrapment efficiency. In vitro release of bacterin Formulation B (4:1), composed of high amount of alginate, showed a burst release of 60% in the first day (Figure 3). In contrast, the bacterin release rate was decreased as the amount of alginate decreased. Formulation C (3:1), D (2:1), E (1.5:1) and F (1:1) illustrated a sustained release of 79.46%, 70.23%, 66.13% and 61.82% in 30 days, respectively (Figure 3). The best-fit model for bacterin release from these MPs systems
Figure 2. Percent bacterin entrapment efficacy prepared with different ratio of alginate and bacterin (Data are presented as mean SEM, n ¼ 3).
was the Higuchi square root model. Furthermore, after 15-day incubation, MPs examined by SEM showed a larger particle size with porous at surface as compared to those of freshly preparation (Figure 4).
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In vivo safety and potency test
Figure 3. Release profiles of bacterin from MPs prepared with different ratio of alginate and bacterin in 0.2 M HEPES buffer, pH 7.4. (^) 4:1; (g) 3:1; (m) 2:1; () 1.5:1 and (–) 1:1. (Data are presented as mean SEM, n ¼ 3).
Formulation E (1.5:1) was selected for further in vivo safety and potency studies. It had water-like consistency with a viscosity of 1.55 cP, while oil adjuvant vaccine had higher viscosity of 25.59 cP. After vaccination, all mice showed no visible unfavorable signs such as necrosis, granuloma or skin sloughing at the site of injection. Then, mice were daily observed. They were alive and showed normal behavior. In addition, vaccine sterility was tested and proved that it was free from any live microbial contaminants (data not shown). The vaccine potency was presented as log protection value which was the difference between log LD50 values of immunized and control mice (Table 2). According to the standard for vaccine production, World Organization for Animal Health (OIE), the difference between LD50 values for immunized and control mice should be at least 4 log units. The MPs vaccine was sufficiently protective in mice. Mice in group I showed log protection of 5.3, while those received 2.5 times of bacterin-loaded MPs, group II, showed slightly increased in log protection of 6. In addition, oil
Figure 4. Scanning electron micrographs of P. multocida-loaded MPs after incubated in 0.2 M HEPES buffer, pH 7.4 for 0 day and 15 days. Scale bar ¼ 10 mm.
P. multocida-loaded alginate microparticles
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Table 2. The potency of MPs vaccine in mice. Group 1 2 3 4
Formulation E (1.5:1) E (1.5:1) Oil adjuvant vaccine (Positive control) Control group (not inoculated)
Route
Cells 108/ml
Viscosity (cP)
IP, 0.2 ml IP, 0.5 ml IP, 0.2 ml –
1.55 1.55 25.59 ND
LD50 2.7
1.04 2.59 1.22 –
10 102.0 101.46 108
Log protection 5.3 6.0 6.54 –
IP, intraperitoneal; ND, not determine. Table 3. Physicochemical stability and potency test of MPs formulation E (1.5:1) after 6 months storage at 4 C and room temperature. 6 months
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Characterization Physicochemical properties D50 (mm) SD Zeta potential (mV) SD Bacterin content (Cells 108/ml) SD Potency test Log protection
0 month
4 C
Room temperature
5.83 0.21 23.07 1.70 4.70 0.15
5.67 0.31 29.35 0.54 4.23 0.18
5.93 0.31 29.57 0.30 4.29 0.30
5.33
4.63
4.33
adjuvant vaccine, a positive control, gave slightly higher log protection of 6.54. Stability test Formulation E (1.5:1) was kept at 4 C and room temperature. After 6 months storage at both conditions (Table 3), the mean particle size and zeta potential were practically unchanged of approximately 6 mm and 30 mV, respectively. In addition, MPs showed percent bacterin remaining of 90%. For in vivo potency test, the protection level of 4.63 and 4.33 log units in mice was observed after 6 month storage at 4 C and room temperature, respectively (Table 3).
Discussion The P. multocida-loaded MPs were successfully prepared by an emulsification-cross-linking technique using calcium as a crosslinking agent. We found that the mean particles size could be successfully reduced to less than 10 mm using a simple mixer and ultrasonic probe. In this technique, both the emulsification and cross-linking steps are crucial for desired particle size. First, water in oil (w/o) emulsion was prepared with the aid of mixer by dispersing alginate solution into oil phase. Then, the internal droplets were further reduced using ultrasonic probe during cross-linking step. Alginate MPs were spontaneously formed by ionic cross-linking using calcium ions. The developed MPs possessed similar mean particle size of 6 mm and zeta potential of 23 mV suggesting that alginate carboxylic group presented on the particle surface. The mean particle size and surface properties of MPs are crucial factors determining the uptake by antigen presenting cells (APCs) such as macrophage and dendritic cells. It is well-known that particles less than 10 mm are best taken up by APCs19–22. Moreover, either negatively or positively charged particles could enhance the interaction of MPs with immune cells, in comparison with neutral charged ones, leading to better taken up by APCs23,24. Based on this, MPs developed in this study showed high potential to be taken up by APCs to stimulate the immune response. The entrapment efficiency and the in vitro bacterin released profile of MPs were found to be dependent on the amount of alginate and calcium chloride. The added calcium ions are commonly known to bind preferentially at the guluronic acid unit of alginate to produce the ‘‘egg-box’’ structure. Therefore, the
density of the matrix, which affects the bacterin entrapment efficiency and release profile, is dependent on the concentration of both alginate and calcium ions. In this study, the amount of calcium ion was kept constant, while the amount of alginate was varied. Therefore, MPs prepared with a lower amount of alginate were more extensively cross-linked with calcium ion producing a denser matrix MPs7,12. Subsequently, this would lead to higher entrapment efficacy and slower released rate than MPs prepared with higher amount of alginate. In vitro dissolution studies revealed that the release kinetic of prepared MPs was best fitted with Higuchi’s model suggesting bacterin released from MPs by diffusion through liquid filled channels of the matrix. As discussed before, the higher amount of alginate resulted in a looser matrix formation. Therefore, a higher amount (60%) of bacterin was released in the first hour for the formulation B (4:1). Initial release decreased with increasing amounts of alginate in the formulation. Formulation C (3:1), D (2:1), E (1.5:1) and F (1:1) released 30, 20, 18 and 12% in the first hour, respectively. This result was in consistent with SEM micrographs. After 15-day dissolution in HEPES buffer, SEM micrograph of formulation B (4:1) showed the most particle erosion with a highly porosity compared to those prepared with lower amount of alginate, formulation C (3:1), D (2:1), E (1.5:1) and F (1:1). It is possible that the observed effect was due to the sodium salt of HEPES, a monovalent cation, may interact with the particles by competing with calcium and resulting in the solubilization of alginate molecules25,26. Thus, the degree of porosity, reflecting on the release rate, depends on the amount of alginate. In addition, a sustained released profile over a period of 30 days was observed. This could permit sustained delivery of antigen which usually translates into a high immune response in vivo27. Formulation E (1.5:1) was selected for the in vitro safety and efficacy studies. It was proved to be safe for parenteral administration in mice. This could be attributed to the advantage that alginate is biodegradable and biocompatible. Furthermore, it was also effective in inducing an immune response in mice. This finding was in agreement with Kidane et al.11 and Suckow et al.13 who found that alginate MPs could induce protection in mice and rabbit, respectively. Particulate vaccine could enhance phagocytosis by APCs and regulate antigen release required for immunization19,28. Moreover, higher amount of bacterin showed a slightly higher log protection value. This could be attributed
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to the fact that high amount of bacterin induce better immune response. Although alginate MPs provided efficient protection, oil adjuvant vaccine gave slightly better protection. This might be due to oil adjuvant vaccine produces depot effect resulting in the slow release of bacterin from the injection site and provides a continuous source of antigen for antibody production over a long period of time. Nevertheless, high viscosity oil adjuvant, 25.59 cP, leads to difficulty for administration and adverse reaction as granuloma is commonly occurred at the injection site. Although this skin reaction can disappear after few months, skin eruption might be observed in some animals29–32. In contrast, the prepared alginate MPs vaccine possessed low viscosity of 1.55 cP which preferable in a large field or farm animals because it is easily injectable through subcutaneous route and does not produce local swelling at site of injection33. In addition, the developed alginate MPs vaccine showed physical and chemical stable over 6 month storage. This was corresponded with the potency test with protection level of 4.5 log unit observed in mice. This vaccine could be kept at both in refrigerator or room temperature which are very versatile in field user.
Conclusions This study showed that P. multocida-loaded alginate MPs for subcutaneous vaccination was successfully developed by emulsification-cross-linking technique. A mean particle size of 6 mm has been successfully developed using simple mixer and ultrasonic probe. The zeta potential of the MPs showed surface negative charge. The bacterin entrapment efficacy of up to 70% was achieved. The slow release of bacterin from MPs was observed up to a 30 day period. The optimized MPs formulation proved to be stable in terms of physical, chemical and potency test. However, further studies are required to investigate the MPs vaccine in cattle and buffalo. Hopefully, it can be applied to farm animals and possibly to undertake the transition to full-scale production and marketing of the final product.
Acknowledgements We acknowledge Faculty of Pharmaceutical Sciences, Naresuan University for providing necessary facilities.
Declaration of interest Financial support from the Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand are gratefully acknowledge. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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RESEARCH ARTICLE
Development of Pasteurella multocida-loaded microparticles for hemorrhagic septicemia vaccine Drug Dev Ind Pharm Downloaded from informahealthcare.com by York University Libraries on 01/01/15 For personal use only.
Pataranapa Nimtrakul1,2, Ratchanee Atthi3, Nanteetip Limpeanchob4, and Waree Tiyaboonchai1,2 1
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand, 2Center of Excellence for Innovation in Chemistry, Commission on Higher Education, Bangkok, Thailand, 3Department of Livestock Development, Bureau of Veterinary Biologics, Pakchong, Nakhonratchasima, Thailand, and 4Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand Abstract
Keywords
In this study, Pasteurella multocida-loaded alginate microparticles (MPs) for subcutaneous vaccination was developed by emulsification-cross-linking technique. Formulation parameter was varied as a ratio of polymer and bacterin. Optical microscopy revealed spherical particles with uniformly distribution. A mean particle size of approximately 6 mm has been successfully constructed using simple mixer and ultrasonic probe. The zeta potential of the MPs showed negatively charge of approximately 23 mV determined by Zeta PalsÕ analyzer. The entrapment efficiency and the in vitro bacterin released profile could be controlled by varying the amount of alginate. The high entrapment efficiency up to 69% was achieved with low concentration of alginate. The MPs possessed a slow bacterin release profile, up to 30 days. In vivo safety and potency tests were proved that the alginate MPs were safe and induced protective immunity in mice. In addition, after storage for 6 months at either 4 C or room temperature, the protective immunity in mice was maintained.
Alginate, hemorrhagic septicemia, microparticles, Pasteurella multocida, vaccine
Introduction Hemorrhagic septicemia (HS) caused by infection with Pasteurella multocida (P. multocida) is a commonly fatal systemic disease of cattle and buffaloes. The disease is prevalent in South and Southeast Asia, serotype B: 2 and in Tropical Africa, serotype E: 21. An outbreak of HS, usually acute, widespread and virulently fatal, would cause economical damage and threaten the cattle export industry. To date, vaccination is the most effective means to prevent P. multocida outbreak. Alum precipitated vaccines (APVs), inactivated vaccine, is the most widely used as it is simple to manufacture. However, APVs provide short term protection, 6 months and post-vaccination shock has been reported up to 10%. In contrast, oil adjuvant vaccines provide longer protection for 1 year. However, these vaccines have disadvantages of high viscosity and post-vaccination high fever in cattle, which make it unpopular among the field users. Some attempts have been done to reduce formulation viscosity, but such vaccines suffer from high number of inactivated cells (1010– 1011)2,3. To overcome those problems, it was developed a new vaccine delivery system possesses lower viscosity, leading to lower side effects during and after injection. Microencapsulation is a unique way to deliver antigens and facilitates their uptake into lymphoid tissue. One of the most
Address for correspondence: Waree Tiyaboonchai, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand. Tel:+66-55-961873. E-mail: [email protected]
History Received 15 August 2013 Revised 27 November 2013 Accepted 2 December 2013 Published online 31 December 2013
common materials used to encapsulate antigens is alginate. It is generally regarded as safe by the United State Food and Drug Administration (FDA) and finding widespread application in pharmaceutical industry4–6. Alginate is a biodegradable, biocompatible and water soluble linear polysaccharide extracted from brown algae. It is composed of alternating block of 1–4 linked a-L-guluronic acid and b-D-mannuronic acid residue7. The parenteral administration of alginate microparticles (MPs) has been shown to provoke immune responses8–13. In the previous study, the alginate MPs were successfully developed by emulsification-cross-linking technique using calcium and zinc as a cross-linking agent14. Unfortunately, a toxicity of this formulation was observed in mice as a result from zinc. Thus, in this study, to improve the safety of MPs, we further developed antigen-loaded MPs using only calcium as a cross-linking agent. The physicochemical properties of MPs were characterized including morphology, mean particle size, zeta potential and entrapment efficiency. The profile of bacterin release was investigated as well as the stability of MPs. Moreover, the safety and efficiency of prepared vaccine was evaluated in vivo.
Materials and methods Materials Sodium alginate, medium viscosity, was purchased from Fluka (Steinheim, UK). PluronicÕ L61 was obtained from BASF (Florham Park, NJ). Olive oil was purchased from TCFF (Bangkok, Thailand). Calcium chloride was purchased from Univar (New South Wales, Australia). Sodium citrate was
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purchased from MERCK (Damstadt, Germany). A formalininactivated whole culture of P. multocida, serotype B:2, 5 was obtained from the Bureau of Veterinary Biologics, Ministry of Agriculture and Cooperatives, Thailand. Micro-BCA protein assay kit was purchased from Thermo Scientific (Rockford, IL). All other chemicals and solvents were of analytical grade.
incubating with 4% w/v sodium citrate. Finally, the amount of bacterin was determined using micro-BCA protein assay kit as described before. The percentage of bacterin released was determined by indirect method according to the equation: Bacterin release ð%Þ 1 Amount of bacterin remained in particle 100 ¼ Initial amount of bacterin
ð2Þ
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Methods Preparation of P. multocida-loaded alginate MPs
In vivo safety and potency test
Pasteurella multocida containing alginate MPs were prepared by emulsification-cross-linking method. The formalin-killed cultures were washed with de-ionized (DI) water before mixed with 2%w/v alginate solution. The alginate mixture was added to oil phase, composed of PluronicÕ L61 in olive oil, while stirred at 1500 rpm using a mixer (RZR 2021, HEIDOLPH Instruments GmbH & Co KG, Germany). The internal droplets were further reduced using ultrasonic probe. Then, cross-linking solution was added drop wise to the emulsion while stirred at 1500 rpm. The resulting antigen MPs were harvested by centrifugation at 1500 rpm for 5 min and washed three times with DI water. Then, MPs were filtrated by a 45 mm sieve. Finally, MPs were adjusted to 40 ml with DI water and kept at 4 C before used.
In vivo experiments were conducted under ethic approval from the Bureau of Veterinary Biologics, Animal Ethics Committee.
Physicochemical characterization of MPs The morphology of both P. multocida-loaded and unloaded microparticles were investigated using scanning electron microscopy (SEM). The mean particle size of the MPs was determined by an optical microscope. At least 300 particles were measured using Feret’s diameter. Cumulative percentage, frequency, undersize and normalized Z-value were calculated. To calculate the geometric mean diameter (D50), particle diameter value were transformed into logarithm value. The zeta potential of the MPs was determined by phase analysis light scattering employing a Zeta PalsÕ (Brookhaven Instruments Corporation, New York). The zeta potential was calculated from the electrophoretic mobility using the Smoluchowski approximation approach15. Determination of bacterin entrapment efficiency MPs were incubated with 4% w/v sodium citrate solution. After dissolution of the particles, samples were centrifuged at 12 000 rpm for 5 min. The resulting bacterin pellets were washed three times with DI water and re-suspended in 1 ml of DI water. Then, the amount of protein referring to bacterin was determined using the bicinchoninic acid (micro-BCA) protein assay in 96-well plates following manufacturer’s instruction. The percent bacterin entrapment efficiency was calculated according to the equation: Bacterin entrapment ð%Þ Amount of bacterin in particle 100 ¼ Initial amount of bacterin
Safety test. The safety test was conducted with acute toxicity testing in mice according to the procedure in compliance with the ASEAN standards of animal vaccines16. Ten male ICR mice, 6–8 weeks of age, were each injected intraperitoneally with 0.5 ml of the sample and were observed daily for 2 weeks. Potency test. The efficiency of MPs vaccines were evaluated by active mouse protection test17. Male ICR mice, 6–8 weeks of age, were equally divided into immunized and control group. Mice in immunized group were inoculated twice with 0.2 ml MPs vaccines by intraperitoneal (IP) route in 2 weeks interval. Mice in control group were not inoculated. One week after the second inoculation, all mice were divided into six subgroups of five mice. Each subgroup was challenged by intraperitoneal inoculation with 0.1 ml of respective dilutions of 6-h broth culture of P. multocida serotype B: 2, 5 in the range of 101 to 106 in immunized group and 104 to 109 in control group. Mice were daily observed for 1 week and lethal dose 50% (LD50) were calculated by the method of Reed & Muench18 according to the equation: Proportional distance Mortality above 50% 50 ¼ Mortality above 50% Mortality below 50%
Log of LD50 ¼ Log of dilution above 50% mortality þ Proportional distance
ð3Þ
ð4Þ
Stability test The stability of MPs in terms of physical and chemical properties was studied both 4 C and room temperature for 6 months. The physical stability of MPs was determined in terms of morphology, particles size, zeta potential and bacterin content as described before. In vivo efficiency studies were determined in terms of potency test.
Results ð1Þ
In vitro release study of bacterin The in vitro release study was performed by keeping samples in the dark at 37 0.5 C for 1 month. Five hundred microliters of MPs were placed in 0.5 ml of 0.2 M HEPES buffer, pH 7.4. At selected time interval (1, 3, 5, 7, 15 and 30 days), the amount of bacterin remained in the MPs were determined. The samples were centrifuged at 3000 rpm for 5 min to collected MPs and remove the released bacterin. Then, MPs were dissolved by
Physicochemical characterization The alginate based MPs were examined by optical microscope and SEM (Figure 1). Optical micrographs revealed that both bacterinloaded and -unloaded MPs were spherical in shape and uniformly distributed. The dark round-like structure seen in the MPs was attributed to the presence of olive oil. Similarly, SEM micrographs of both bacterin-loaded and -unloaded MPs also showed spherical particles with smooth surface. The mean particle size and zeta potential were found to be independent on ratio of polymer and bacterin. All formulations possessed similar mean particle size of approximately 6 mm and zeta potential value of approximately 23 mV (Table 1).
P. multocida-loaded alginate microparticles
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Figure 1. Optical micrographs of formulation E (1.5:1) (A) blank MPs, (B) bacterin-loaded MPs (400 magnifications) and Scanning electron micrographs of formulation E (1.5:1) (C) blank MPs and (D) bacterin-loaded MPs (800 magnifications). Table 1. Mean particle size and zeta potential of MPs prepared with different ratio of alginate and bacterin (n ¼ 3). D50 (mm) SD Formulation A B C D E F
Zeta potential (mV) SD
Ratio of alginate:bacterin
Blank MPs
Bacterin-loaded MPs
Blank MPs
Bacterin-loaded MPs
8:1 4:1 3:1 2:1 1.5:1 1:1
6.50 0.14 6.70 0.10 5.97 0.21 5.80 0.40 5.90 0.10 6.03 0.32
6.10 0.14 6.13 0.49 5.97 0.38 5.97 0.25 5.83 0.21 5.77 0.21
20.39 1.51 22.22 0.87 22.15 0.72 21.70 0.27 23.23 1.05 21.14 0.78
24.21 1.44 23.40 0.51 22.93 0.88 22.78 0.58 23.07 1.70 22.92 1.71
Bacterin entrapment efficiency MPs were prepared by varying the ratio of alginate and bacterin, while the amount of calcium chloride kept constant. As the amount of alginate increased, formulation A (8:1) shows the lowest entrapment efficiency of 15% was observed (Figure 2). In contrast, formulation B (4:1) and C (3:1) showed entrapment efficiency of 43 and 61%, respectively. The high entrapment efficiency up to 69% was observed with formulation D (2:1), E (1.5:1) and F (1:1). Nevertheless, application of hardening, washing and filtering of MPs during production could potentially decrease the entrapment efficiency. In vitro release of bacterin Formulation B (4:1), composed of high amount of alginate, showed a burst release of 60% in the first day (Figure 3). In contrast, the bacterin release rate was decreased as the amount of alginate decreased. Formulation C (3:1), D (2:1), E (1.5:1) and F (1:1) illustrated a sustained release of 79.46%, 70.23%, 66.13% and 61.82% in 30 days, respectively (Figure 3). The best-fit model for bacterin release from these MPs systems
Figure 2. Percent bacterin entrapment efficacy prepared with different ratio of alginate and bacterin (Data are presented as mean SEM, n ¼ 3).
was the Higuchi square root model. Furthermore, after 15-day incubation, MPs examined by SEM showed a larger particle size with porous at surface as compared to those of freshly preparation (Figure 4).
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In vivo safety and potency test
Figure 3. Release profiles of bacterin from MPs prepared with different ratio of alginate and bacterin in 0.2 M HEPES buffer, pH 7.4. (^) 4:1; (g) 3:1; (m) 2:1; () 1.5:1 and (–) 1:1. (Data are presented as mean SEM, n ¼ 3).
Formulation E (1.5:1) was selected for further in vivo safety and potency studies. It had water-like consistency with a viscosity of 1.55 cP, while oil adjuvant vaccine had higher viscosity of 25.59 cP. After vaccination, all mice showed no visible unfavorable signs such as necrosis, granuloma or skin sloughing at the site of injection. Then, mice were daily observed. They were alive and showed normal behavior. In addition, vaccine sterility was tested and proved that it was free from any live microbial contaminants (data not shown). The vaccine potency was presented as log protection value which was the difference between log LD50 values of immunized and control mice (Table 2). According to the standard for vaccine production, World Organization for Animal Health (OIE), the difference between LD50 values for immunized and control mice should be at least 4 log units. The MPs vaccine was sufficiently protective in mice. Mice in group I showed log protection of 5.3, while those received 2.5 times of bacterin-loaded MPs, group II, showed slightly increased in log protection of 6. In addition, oil
Figure 4. Scanning electron micrographs of P. multocida-loaded MPs after incubated in 0.2 M HEPES buffer, pH 7.4 for 0 day and 15 days. Scale bar ¼ 10 mm.
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Table 2. The potency of MPs vaccine in mice. Group 1 2 3 4
Formulation E (1.5:1) E (1.5:1) Oil adjuvant vaccine (Positive control) Control group (not inoculated)
Route
Cells 108/ml
Viscosity (cP)
IP, 0.2 ml IP, 0.5 ml IP, 0.2 ml –
1.55 1.55 25.59 ND
LD50 2.7
1.04 2.59 1.22 –
10 102.0 101.46 108
Log protection 5.3 6.0 6.54 –
IP, intraperitoneal; ND, not determine. Table 3. Physicochemical stability and potency test of MPs formulation E (1.5:1) after 6 months storage at 4 C and room temperature. 6 months
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Characterization Physicochemical properties D50 (mm) SD Zeta potential (mV) SD Bacterin content (Cells 108/ml) SD Potency test Log protection
0 month
4 C
Room temperature
5.83 0.21 23.07 1.70 4.70 0.15
5.67 0.31 29.35 0.54 4.23 0.18
5.93 0.31 29.57 0.30 4.29 0.30
5.33
4.63
4.33
adjuvant vaccine, a positive control, gave slightly higher log protection of 6.54. Stability test Formulation E (1.5:1) was kept at 4 C and room temperature. After 6 months storage at both conditions (Table 3), the mean particle size and zeta potential were practically unchanged of approximately 6 mm and 30 mV, respectively. In addition, MPs showed percent bacterin remaining of 90%. For in vivo potency test, the protection level of 4.63 and 4.33 log units in mice was observed after 6 month storage at 4 C and room temperature, respectively (Table 3).
Discussion The P. multocida-loaded MPs were successfully prepared by an emulsification-cross-linking technique using calcium as a crosslinking agent. We found that the mean particles size could be successfully reduced to less than 10 mm using a simple mixer and ultrasonic probe. In this technique, both the emulsification and cross-linking steps are crucial for desired particle size. First, water in oil (w/o) emulsion was prepared with the aid of mixer by dispersing alginate solution into oil phase. Then, the internal droplets were further reduced using ultrasonic probe during cross-linking step. Alginate MPs were spontaneously formed by ionic cross-linking using calcium ions. The developed MPs possessed similar mean particle size of 6 mm and zeta potential of 23 mV suggesting that alginate carboxylic group presented on the particle surface. The mean particle size and surface properties of MPs are crucial factors determining the uptake by antigen presenting cells (APCs) such as macrophage and dendritic cells. It is well-known that particles less than 10 mm are best taken up by APCs19–22. Moreover, either negatively or positively charged particles could enhance the interaction of MPs with immune cells, in comparison with neutral charged ones, leading to better taken up by APCs23,24. Based on this, MPs developed in this study showed high potential to be taken up by APCs to stimulate the immune response. The entrapment efficiency and the in vitro bacterin released profile of MPs were found to be dependent on the amount of alginate and calcium chloride. The added calcium ions are commonly known to bind preferentially at the guluronic acid unit of alginate to produce the ‘‘egg-box’’ structure. Therefore, the
density of the matrix, which affects the bacterin entrapment efficiency and release profile, is dependent on the concentration of both alginate and calcium ions. In this study, the amount of calcium ion was kept constant, while the amount of alginate was varied. Therefore, MPs prepared with a lower amount of alginate were more extensively cross-linked with calcium ion producing a denser matrix MPs7,12. Subsequently, this would lead to higher entrapment efficacy and slower released rate than MPs prepared with higher amount of alginate. In vitro dissolution studies revealed that the release kinetic of prepared MPs was best fitted with Higuchi’s model suggesting bacterin released from MPs by diffusion through liquid filled channels of the matrix. As discussed before, the higher amount of alginate resulted in a looser matrix formation. Therefore, a higher amount (60%) of bacterin was released in the first hour for the formulation B (4:1). Initial release decreased with increasing amounts of alginate in the formulation. Formulation C (3:1), D (2:1), E (1.5:1) and F (1:1) released 30, 20, 18 and 12% in the first hour, respectively. This result was in consistent with SEM micrographs. After 15-day dissolution in HEPES buffer, SEM micrograph of formulation B (4:1) showed the most particle erosion with a highly porosity compared to those prepared with lower amount of alginate, formulation C (3:1), D (2:1), E (1.5:1) and F (1:1). It is possible that the observed effect was due to the sodium salt of HEPES, a monovalent cation, may interact with the particles by competing with calcium and resulting in the solubilization of alginate molecules25,26. Thus, the degree of porosity, reflecting on the release rate, depends on the amount of alginate. In addition, a sustained released profile over a period of 30 days was observed. This could permit sustained delivery of antigen which usually translates into a high immune response in vivo27. Formulation E (1.5:1) was selected for the in vitro safety and efficacy studies. It was proved to be safe for parenteral administration in mice. This could be attributed to the advantage that alginate is biodegradable and biocompatible. Furthermore, it was also effective in inducing an immune response in mice. This finding was in agreement with Kidane et al.11 and Suckow et al.13 who found that alginate MPs could induce protection in mice and rabbit, respectively. Particulate vaccine could enhance phagocytosis by APCs and regulate antigen release required for immunization19,28. Moreover, higher amount of bacterin showed a slightly higher log protection value. This could be attributed
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to the fact that high amount of bacterin induce better immune response. Although alginate MPs provided efficient protection, oil adjuvant vaccine gave slightly better protection. This might be due to oil adjuvant vaccine produces depot effect resulting in the slow release of bacterin from the injection site and provides a continuous source of antigen for antibody production over a long period of time. Nevertheless, high viscosity oil adjuvant, 25.59 cP, leads to difficulty for administration and adverse reaction as granuloma is commonly occurred at the injection site. Although this skin reaction can disappear after few months, skin eruption might be observed in some animals29–32. In contrast, the prepared alginate MPs vaccine possessed low viscosity of 1.55 cP which preferable in a large field or farm animals because it is easily injectable through subcutaneous route and does not produce local swelling at site of injection33. In addition, the developed alginate MPs vaccine showed physical and chemical stable over 6 month storage. This was corresponded with the potency test with protection level of 4.5 log unit observed in mice. This vaccine could be kept at both in refrigerator or room temperature which are very versatile in field user.
Conclusions This study showed that P. multocida-loaded alginate MPs for subcutaneous vaccination was successfully developed by emulsification-cross-linking technique. A mean particle size of 6 mm has been successfully developed using simple mixer and ultrasonic probe. The zeta potential of the MPs showed surface negative charge. The bacterin entrapment efficacy of up to 70% was achieved. The slow release of bacterin from MPs was observed up to a 30 day period. The optimized MPs formulation proved to be stable in terms of physical, chemical and potency test. However, further studies are required to investigate the MPs vaccine in cattle and buffalo. Hopefully, it can be applied to farm animals and possibly to undertake the transition to full-scale production and marketing of the final product.
Acknowledgements We acknowledge Faculty of Pharmaceutical Sciences, Naresuan University for providing necessary facilities.
Declaration of interest Financial support from the Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand are gratefully acknowledge. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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