Preparation of alginate coated chitosan microparticles for vaccine delivery
Absorption of antigens onto chitosan microparticles via electrostatic interaction is a common and relatively mild process suitable for mucosal vaccine. In order to increase the stability of antigens and prevent an immediate desorption of antigens from chitosan carriers in gastrointestinal tract, coating onto BSA loaded chitosan microparticles with sodium alginate was performed by layer-by-layer technology to meet the requirement of mucosal vaccine.
The prepared alginate coated BSA loaded chitosan microparticles had loading efficiency (LE) of 60% and loading capacity (LC) of 6% with mean diameter of about 1 μm. When the weight ratio of alginate/chitosan microparticles was greater than 2, the stable system could be obtained. The rapid charge inversion of BSA loaded chitosan microparticles (from +27 mv to -27.8 mv) was observed during the coating procedure which indicated the presence of alginate layer on the chitosan microparticles surfaces. According to the results obtained by scanning electron microscopy (SEM), the core-shell structure of BSA loaded chitosan microparticles was observed. Meanwhile, in vitro release study indicated that the initial burst release of BSA from alginate coated chitosan microparticles was lower than that observed from uncoated chitosan microparticles (40% in 8 h vs. about 84% in 0.5 h). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) assay showed that alginate coating onto chitosan microparticles could effectively protect the BSA from degradation or hydrolysis in acidic condition for at least 2 h. The structural integrity of alginate modified chitosan microparticles incubated in PBS for 24 h was investigated by FTIR.
The prepared alginate coated chitosan microparticles, with mean diameter of about 1 μm, was suitable for oral mucosal vaccine. Moreover, alginate coating onto the surface of chitosan microparticles could modulate the release behavior of BSA from alginate coated chitosan microparticles and could effectively protect model protein (BSA) from degradation in acidic medium in vitro for at least 2 h. In all, the prepared alginate coated chitosan microparticles might be an effective vehicle for oral administration of antigens.
KeywordsChitosan Bovine Serum Albumin Alginate Zeta Potential Sodium Alginate
bovine serum albumin
scanning electron microscopy
SDS-polyacrylamide gel electrophoresis
Fourier transform infra-red
Development of an oral antigens (protein, and etc) delivery system for mucosal vaccine is a meaningful challenge for pharmaceutical scientists. The instability and poor absorption of antigens in gastrointestinal tract is major obstacles in the development of oral antigen delivery system for mucosal vaccine. Problems such as acid degradation in stomach, poor permeability across the gastrointestinal mucosa and the first-pass metabolism greatly limited the uptake of antigens by M-cell which is very important step for immune response [1, 2]. To overcome the above-mentioned obstacles, several strategies, including liposomes [3, 4, 5], micro/nanoparticles [6, 7, 8], micro/nanoemulsion , and etc, have been explored to encapsulate antigens for the mucosal vaccine. Among these strategies, micro/nanoparticles made of biodegradable natural polymer have gained considerable interest in the past decades. One important aspect is that some natural polymers, especially chitosan, haves been demonstrated that could enhance the immunogenicity of poor immune response antigens in the form of solution and micro/nanoparticles [10, 11].
Chitosan, as a cationic polysaccharide, has gained increasing attention in pharmaceutical field due to its favorable biological properties, such as non-toxicity, biodegradability [1, 12], mucoadhesive properties [13, 14], and etc. Additionally, chitosan micro/nanoparticles can be easily prepared by ionic gelation method using tripolyphosphate (TPP) as precipitating agent [12, 15]. The advantage of this method was attributed to the mild condition without the application of harmful organic solvent at room temperature in the procedure, and also could efficiently detain the bioactivity of macromolecules (protein, DNA etc) during the encapsulation. In spite of all its superior properties, chitosan has an apparent pKa of 5.6 and is only soluble in acidic solutions. When incubated in physiological fluid environment, chitosan will lose its capacity of mucoadhesive properties and permeation enhancing effect due to the deprotonation of chitosan, which would make chitosan carriers lose its advantage compared with other carriers for mucosal vaccine. Meanwhile, chitosan has limited ability for controlling the release of encapsulated macromolecule compounds because of its hydrophilic nature and easy solubility in acidic medium [1, 16]. It might be an interesting method to overcome these obstacles by coating acid-resistant polymer, such as alginate sodium, onto the surface of chitosan microparticles. As an anionic polysaccharide with favorable biological properties, alginate can easily interact with cationic chitosan microparticles to form the polyelectrolyte complex via electrostatic interactions [3, 17, 18, 19]. Additionally, this coating procedure was performed at relatively mild condition without using any organic solvent. This relatively mild process has enabled not only proteins, but cells and DNA to be incorporated into the chitosan/alginate matrices with retention of biological activity .
In this work, we hope to develop a novel oral antigen carrier based on alginate coated chitosan microparticles to meet the requirement of mucosal vaccine. Model protein (BSA) was adopted to evaluate the properties of alginate coated chitosan microparticles. In vitro release behavior of BSA from chitosan microparticles and the stability of BSA loaded chitosan microparticles against acidic condition could be modified by alginate coating layer.
Chitosan with deacetylation (DA) of 92% and viscosity of 55 mpa.s (1% in 1% acetic acid, 20°C) was provided by Sigma (USA). Sodium alginate with viscosity of 20–40 cp (1% in distilled water) was obtained from Aldrich (USA). Bovine serum albumin (BSA) was purchased from BoAo Biochemical Company (Shanghai, China). Sodium tripolyphosphate was bought from Sigma (USA), and CaCl2 was bought from Chengdu KeLong Chemicals (Chengdu, China). BCA™ kit was provided by Pierce (USA). All other chemicals used in this paper were agent grade. Ultrapure water from Milli-Q water system was used to prepare the aqueous solutions.
Preparation of chitosan microparticles
Chitosan microparticles were prepared by the ionic gelation of chitosan solution with anionic tripolyphosphate (TPP). Briefly, chitosan was dissolved in 1% (v/v) acetic acid aqueous solution at concentration of 5 mg/ml. Then, TPP was dissolved in distilled water at the concentration of 1 mg/ml. Subsequently, 9 ml of TPP solution was added dropwisely into 18 ml of chitosan solution (5 mg/ml), chitosan colloid microparticles were formed spontaneously under mild agitation at room temperature. Ten minutes later, chitosan colloid microparticles were centrifuged (Beckman Coulter™, Avanti™ J-30I centrifuge, Germany) at 9,500 rpm for 15 min. Then, the supernatant was discarded and the deposit was re-dispersed in distilled water for further use.
Loading bovine serum albumin (BSA) to chitosan microparticles
Colloid chitosan microparticles were re-dispersed in 25 ml of distilled water at concentration of 5 mg/ml under continuous ultrasonication (Benchtop 20L, Medisafe, UK Ltd, UK) to disaggregate the chitosan microparticles. The loading procedure was performed by incubating different concentrations of BSA with chitosan microparticles under mild agitation at room temperature for 15 min. Loading efficiency (LE) and loading capacity (LC) of BSA on chitosan microparticles were detected in an indirect way by determining the free BSA remained in the supernatant after the performance of centrifuge, and the method was shown as following. One milliliter of BSA loaded chitosan microparticles suspension was centrifuged (Centrifuge 5415D, Eppendorf, Germany) at 13,200 rpm for 20 min and the amount of BSA in the supernatant was measured by BCA™ kit . The supernatant of blank chitosan microparticles was adopted as the blank to correct the absorbance reading value of the BSA-loaded chitosan microparticles. The corrected optical density (OD) value was then used to calculate the concentration of BSA in the supernatant.
Preparation of alginate coated chitosan microparticles
BSA loaded chitosan microparticles suspensions with pH value at 5.1 were added dropwisely into sodium alginate solution (pH = 7.2) at concentration of 10 mg/ml under mild agitation for 10 min. Then the suspension was centrifuged at 3,400 rpm for 5 min, and the supernatant was discarded. Finally, alginate coated chitosan microparticles were re-dispersed into calcium chloride (CaCl2) aqueous solution (pH = 7.0) at concentration of 0.524 mmol/L to crosslink the alginate layer presents on the surface of chitosan microparticles.
Morphological characterization, size and surface charge
The morphological characteristics of microparticles were examined by scanning electron microscopy (JSM-5900LV, JEOL, Japan). Microparticles were sputtered with gold and maintained at room temperature for complete dryness before the observation.
The particle size distribution was detected by laser diffraction (Nano-ZS 90, Malvern Instrument, UK; BT-2002 Laser Particle Size Analyzer, Dandong Bettersize Instruments LTD, China). The zeta potential of particles was examined by Malvern Zeta analyzer (Nano-ZS 90, Malvern Instrument, UK) with ultrapure water as solvent (pH = 7, 25°C). These measurements were run at least three times with independent particle batches.
Protein release in vitro
In vitro release behavior of BSA from uncoated and alginate coated chitosan microparticles were determined as followed. One milliliter of microparticles suspension was first centrifuged and the deposit was incubated in 1 ml of phosphate buffer saline (PBS, pH7.4) in Eppendorf tube (EP tube). Then, the EP tube was placed in an air shaker bath at 100 rpm/min (at 37°C) for in vitro release. At scheduled time, samples were centrifuged at 13,200 rpm for 20 min and the supernatant was replaced with fresh PBS (pre-warmed to 37°C). The amount of BSA presented in the supernatant was determined by BCA™ kit as described in section 2.3. According to protocol, the amount of BSA released was expressed as a percentage of total BSA encapsulated in chitosan microparticles as calculated from the LE value.In vitro release experiments were repeated three times.
Acidic degradation protection assay
Different formulations of chitosan microparticles (BSA loaded chitosan microparticles and alginate coated BSA loaded chitosan microparticles) were first centrifuged at 13,000 rpm for 20 min and the supernatant was discarded. Then, the deposition was incubated with 0.5 ml of HCl (0.01 M) in air shaker bath at 37°C for 2 h. Finally, the reaction was stopped by 0.5 ml of aqueous NaOH (0.01 M) solution. These systems were sustained release for another 24 h with addition of PBS to final volume at 4 ml. Twenty four hours later, the supernatant containing released BSA was collected and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The pure BSA solution was designed as the control.
Fourier transform infra-red (FTIR) measurements
FTIR measurements were taken at room temperature using NICOLET 200SXV Infrared Spectrophotometer (USA). Alginate coated chitosan microparticles at 0 h and 24 h of release test in PBS (pH = 7.4) were centrifuged and washed with ultrapure water, finally freeze-dried overnight before the detection.
Results and discussion
Preparation of alginate coated BSA loaded chitosan microparticles
The size and zeta potential of alginate/BSA/chitosan system
Mean particles size (nm)
Zeta potential (mV)
Blank chitosan microparticles a
BSA-loaded chitosan particles b
Alginate coated BSA loaded chitosan microparticles c
Effect of alginate/chitosan microparticles weight ratio on the properties of alginate -chitosan microparticles formulation
Alginate/chitosan microparticles weight ratio
Mean particles size (nm)
2722 (partially precipitation)
Procedure of protein loading
Effect of BSA concentration
The mean particles size and zeta potential of BSA loaded chitosan microparticles
BSA concentration (mg/ml)
Mean particle size (nm)
Zeta potential (mV)
Chitosan microparticles at concentration of 5 mg/ml
Effect of alginate/chitosan microparticles weight ratio
Characterization of chitosan microparticles
Protein release in vitro
Acidic degradation protection
Fourier transform infra-red (FTIR) measurements
The prepared alginate coated chitosan microparticles, with mean diameter of about 1 μm, was suitable for oral administration. Moreover, alginate coating onto surface of chitosan microparticles could modulate the release behavior of BSA from alginate coated chitosan microparticles and could effectively protect model protein (BSA) from degradation against acidic medium (pH2) in vitro at least for 2 hours. According to FTIR, some alginate on surface of chitosan microparticles at 24 h of release test has been dissolved into PBS. Based on the information demonstrated, the prepared alginate coated chitosan microparticles might be an effective vehicle for oral administration of antigens.
This work was financially supported by National 863 project (2007AA021902), National Natural Science Foundation of China (NSFC20704027), Sichuan Key Project of Science and Technology (2007SGY019), Sichuan Prominent Young Talents Program (07ZQ026-033), and Chinese Key Basic Research Program (2004CB518807).
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