key: cord-0970747-xoowaowx
authors: Sun, Qingshen; Han, Dequan; Lei, Hong; Zhao, Kai; Zhu, Li; Li, Xiaodi; Fu, Honggang
title: Preparation and characterization of chitosan microsphere loading bovine serum albumin
date: 2012-06-09
journal: J Wuhan Univ Technol Mater Sci Ed
DOI: 10.1007/s11595-012-0485-4
sha: 59a49ad48e21f54f70ae17461581d795a7707d36
doc_id: 970747
cord_uid: xoowaowx

To optimize the preparation process of chitosan microspheres and study its loading capacity, chitosan microsphere was prepared by crosslinking with glutaraldehyde, and bovine serum albumin (BSA) was absorbed onto chitosan microsphere. Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FITR), TA instruments and zeta potentiometer analyzer were used to characterize the parameters with respect to size, thermal characters, morphology, and zeta potential of the microspheres. The loading capability and in vitro release tests were carried out. The results showed that chitosan microsphere with particle size less than 10 μm and positively charged (+25.97±0.56 mV) can be obtained under the aldehyde group to amino group ratio at 1:1. A loading capacity of BSA at 28.63±0.15 g/100 g with corresponding loading efficiency at 72.01±1.44% was obtained for chitosan microsphere. In vitro test revealed a burst release followed by sustained-release profile.

In encapsulating processes, the drugs may release through different mechanisms, such as diffusion. The residue drugs on the surface of these microcapsules may account for the burst-release stages. Another type of sustained-release drug delivery system (DDS) is that the objective molecules were absorbed onto the surfaces of the microcapsules by electrostatic interactions or hydrogen bonds. The common used polymers in this system include poly(lactic acid) (PLA), poly(D,Llactic-co-glycolic acid) (PLGA) [1] , zein [2] , chitosan, and so on. All these substances have the advantages of good biodegradability and biocompatibilities. However, PLA or PLGA may undergo bulk erosion in vivo that produces a very low pH environment within these matrices, which may adversely affect sensitive therapeutic agents such as proteins. Zein is a natural polymer, which has many advantages as carrier for DDS [2] , but these protein suffers from its sources, as the extraction of zein from corn is a laborious process [3] .

Chitosan is the only natural alkaline polysaccharide containing many free amino groups. This polysaccharide has such features as non-toxic, biodegradable, and biocompatibility, which is obtained from deacetylation of chitin, and is a linear chain polysaccharide formed through the linkage of α-amino-D-glucosamine by β-1,4-glucosidic bond. Chitosan with high molecular weight is insoluble in water and organic solvents, but can dissolve in weak acids by converting the glucosamine units into R-NH 3 + [4, 5] . As many amino and hydroxyl groups distributed on the chains of chitosan molecules, the inner-or inter-hydrogen bonds can be formed. Chitosan has many bioactivities such as immunity, antibiosis, and wound healing, and has been used in waste water disposal [6] [7] [8] , food industry, spinning, chemical industry, bioengineering, drug delivery system [4, [9] [10] [11] and tissue engineering [12] as a kind of functional polymer. Chitosan has also been used in agriculture as sustained-release carrier for fertilizer in the form of microparticles [13] . Chitosan has been used not only in the natural form, but also in the form of microcapsule, as microcapsule can be used as carriers for sustained release of drugs or other active molecules. The aim of this paper is to use chitosan microsphere adsorping BSA as model in DDS.

Chitosan microspheres/nanoparticles have been prepared by different methods such as ioncrosslinking, ionic/covalent crosslinking [14] [15] [16] , chemical crosslinking [17] , membrane emulsifi cation technique [18] , spray-drying technique [19] , inverse phase emulsion dispersion method [20] , compounding strategies such as modified ionotropic gelation method combined with a high voltage electrostatic field [21] and there are many reports on the uses of chitosan microspheres or nanoparticles as carriers as protein [15, 16, [21] [22] [23] [24] [25] [26] [27] [28] , and DNA delivery systems [24] . In addition, the modifi ed chitosan hydrogel with pH-sensitive characteristics has also been developed for controlling the release of protein molecules [23, 29, 30] . In the protein delivery system, bovine serum albumin (BSA) is the most frequently used model protein molecules [19, 30, 31] . Recently, the chitosan-BSA system has been designed as model for isolation of protein from the solution as this system does not change the secondary or tertiary structure of the protein molecules [32] . The sustained-or targetted-drug delivery system based on chitosan and other polymers such as alginate and pectin has also been attempted [22, 25, 28, [33] [34] [35] [36] . However, less study has been conducted on the relationship between the combining ways of the BSA on chitosan microspheres and their relationship to the loading capacity and in vitro release profi le. Therefore, the purpose of this study is to elucidate the relationship between the surface features of chitosan microspheres and their loading capacity to BSA. The possible absorptive mechanisms of chitosan microsphere were indicated.

Chitosan with≥90% deacetylation degree was purchased from Jinan Haibeide Co., Ltd. (Shandong, China), and the viscosity-average molecular weight is 466 KD. BSA was purchased from Shanghai Pufei Bio-Technology Co., Ltd. Bradford reagent was supplied by Applygen Technologies Inc.

Chitosan microspheres were prepared by chemical crosslinking method. Briefl y, a 1% chitosan solution (w/ v) was dissolved thoroughly into an aqueous solution of acetic acid (2% v/v) under stirring at 12 000 rpm. 2 g span 80 was added into 100 mL liquid paraffi n to form 2% solution (w/v). Then 200 mL chitosan solution was added drop wise (about 1 mL/min) into the liquid paraffin under magnetic stirring to form water-in-oil (w/o) emulsion. After adding the chitosan solution, the stirring was continued for another 10 min. Different amount of glutaraldehyde was added into the solution as crosslinker with the final ratio of aldehyde group to amino group at 1:1, and the solution was stirred for another 3 h. The microparticle suspension was subsequently centrifuged at 4 000 rpm for 25 min. The pellet was collected and washed in turn with 10 mL petroleum, isopropyl alcohol and water, respectively. This washing procedure was repeated triplicate before freeze-drying of the pellet overnight. Lyophillization of the samples was performed using a Christ freeze-dryer.

For the preparation of chitosan microsphere containing BSA, the adsorption process was employed as reported previously [31] , in which the blank microsphere and 0.5 mg/mL BSA in pH 5.8 PBS were prepared. The chitosan microsphere was added into the BSA solution with chitosan/BSA ratio at 2:1 (w/w) and incubated at 4 ℃ for 24 h. The solution was centrifuged at 4 000 rpm for 20 min and washed with water for three times. The pellets were lyophilized.

Microstructure analysis of the microspheres prepared by different protocols were carried out by mounting the microsphere onto SEM stubs, and sputter coated with gold for 3 min for SEM analysis (S-3400, Hitachi science systems, Ltd) at an accelerating voltage of 10 kV. FTIR analysis was conducted on an FT-IR spectrometer (spectrum one, Perkin Elmer, Ltd, America) to characterize chemical functional groups of chitosan microsphere with different preparation conditions and the effect of BSA adsorption on the changes of chitosan surfaces. 5-10 mg powder samples were compressed into KBr disks for the FT-IR measurement. The spectrum was collected in a range between 4 000 and 370 cm 1 with a resolution of 1 cm 1 (100 scans per sample). The dried chitosan microsphere was suspended into deionized water at a certain concentration. The size distribution was determined by measuring the size of microspheres from SEM images, 10 microspheres were selected randomly from each image and the sizes were averaged. The zeta potential of the chitosan microspheres was measured in demineralized water at neutral pH by dynamic light scattering (DLS) with a Zetasizer 3000 HS instrument (Malvern Instruments, Malvern, UK).

Thermal properties of the different materials were analyzed by thermogravimetric analysis (TGA) by DSC-TGA Standard Module. 5-10 mg samples were measured on a Perkin Elmer Pyris Diamond TG/DTA analyzer at a heating rate of 10 ℃ min 1 under nitrogen atmosphere over the temperature range of 37-650 ℃.

Samples of approximately 10-20 mg were used for the TGA measurements. 2.3 Estimation of BSA content in the chitosan microspheres prepared by different process BCA protein assay kit was used to determine the loading efficiency of BSA in chitosan microsphere according to the instructions of manufacturer. In the adsorption process, the free BSA was determined after centrifugation at 4 000 rpm for 20 min, the loading efficiency (LE) and loading capacity (LC) were calculated according to the following formula:

(1)

(2)

In vitro release experiments for drug-loaded microparticles were performed in pH7.4 PBS buffer. Before the experiments, the release medium was prepared and placed for equilibrium at 37 ℃ overnight. The 10 mg microparticles were then placed in different beakers containing 50 mL release medium at 37± 0.5 ℃. 3 mL aliquots were withdrawn at 0.5, 1, 2, 4, 8, 24 hours post incubation for the first 24 hours, and then at 24 h intervals for the following periods and replaced with an equal volume of fresh medium throughout the study. The BSA release amount were assayed by measuring absorbance value at 280 nm. The accumulative release rate of BSA was calculated. The release experiments were performed triplicate and the results were expressed as ±Std.

All the tests were repeated at least in triplicates, and the results were expressed as ± Std,

In the drug or protein delivery systems, the microspheres prepared from biodegradable polymers such as chitosan, PLA have been used extensively [36] . The possible adsorption ways of BSA on the surfaces of chitosan microspheres is studied in this work. Chemical crosslinking method was used for the preparation of chitosan microsphere in this study. The hypothesis was that the BSA molecules adhered to different locus of the chitosan microsphere surface, as the surface was not absolutely smooth, or the amino groups were not crosslinked by glutaraldehyde molecules completely, the BSA molecules combined at these loci with different interactions, that is, physical adhesion or electrostatic interactions.

The morphology of the presented chitosan microspheres was analyzed by SEM as shown in Fig.1 . The chitosan microspheres with spherical appearance can be obtained. The zeta potential was positive (25.97 ±0.56 mV) when the aldehyde to amino ratio at 1:1, which suggested the positive charges of the chitosan microsphere. Higher zeta potential also means good dispersibility of these microspheres.

The blank chitosan microsphere crosslinked by glutaraldehyde with different aldehyde group and amino group ratio at 1:1; 3:1; 5:1; 10:1 and 20:1 for as many as five batches were prepared. The result showed that the morphology and uniform of chitosan microspheres changed significantly with the increasing value of glutaradehyde to amino ratio (data not shown). The optimal aldehyde to amino ratio is 1:1 for the preparation of blank microsphere in terms of appearance and uniformity (Fig.1) . When the aldehyde/amino ratio increased to 20:1, the chitosan microspheres sticked to each other, which is not expected as it is assumed that higher content of aldehydes may cause the amino groups to crosslink more thoroughly, thus become more regular in the appearance of the microsphere. High aldehyde/amino ratio need less time to form microspheres due to the increased cross-linking rate. So under the same crosslinking time, the whole microspheres which had formed in the high aldehyde/amino ratio reaction system were broken into pieces under the shearing force of solution.

3.2 FT-IR analysis of the group changes F i g . 2 i s t h e F T I R s p e c t r u m o f c h i t o s a n microspheres. In this study dried drug-loaded and blank chitosan microspheres were analyzed by FTIR to observe the possible interaction of the functional groups of all the molecules. Chitosan powder (a) exhibits main characteristic bands of hydroxide group (-OH) at 3 435.02 cm 1 . The broad peaks at 1 646 cm 1 and 1 539 cm 1 is stretching vibration of amide I and II bands, respectively, which indicated signifi cant deacetylation of the chitosan molecule. After crosslinking between chitosan and glutaradehyde (b), the two peaks were located at 1 557 cm 1 and 1 654 cm 1 , respectively. The significant decreased transmittance at 1 638 cm 1 as shown in Fig.2(d) , which is assigned to the vibrations of amide linkages, confirmed the formation of new chemical bonds between the residue amino groups of chitosan microspheres and the carbonyl groups of BSA molecules, which may cause the increase of TG 50 as shown below. The wider peak appeared at 3 435.02 cm 1 may be explained by the accumulative effects of the -OH vibrations from both chitosan and BSA molecules as shown in Fig.2(d) .

TG analysis results were shown in Fig.3 . The temperature at which 50% weight loss (TG 50 ) was 318.55°, 364.92°, 391.65° and 394.81°, respectively for chitosan powder, chitosan microsphere, BSA and chitosan microsphere bearing BSA, respectively. The significant higher TG 50 of chitosan microspheres compared with that of chitosan powder indicates the amino groups of chitosan have been crosslinked by the aldehyde groups. In addition, chitosan microsphere bearing BSA has highest TG 50 , which also indicated that in the adsorption process, the BSA molecules acts not only as the objective molecules, but also act as partial crosslinking effects.

The results of UV/Vis spectroscopy showed that the concentrations (X) and the absorbance (Y) of BSA had good linear relationship. The linear equation is y=1.6229x-0.0035, R=0.9999. To ensure that the absorbed BSA released completely from the chitosan microsphere, the loaded microspheres were dissolved in hydrochloric acid at pH 2. After 2 h of incubation, the chitosan microparticles were found to be completely disintegrated and the BSA in the suspension could be determined for loading effi ciency and loading capacity. A loading capacity 28.63±0.15 g/100 g with corresponding loading efficiency at 72.01±1.44% was obtained for chitosan microsphere, which reached the highest encapsulation efficiency of chitosan/TPP nanoparticles as prepared by Yang et al [15] . However, this value is lower than the proteinloaded chitosan microspheres which is prepared by a modified ionotropic gelation method combined with a high voltage electrostatic field which showed prospective method for the preparation of proteindelivery system [21] . Fig.4 showed an obvious burst release was observed for chitosan microspheres which is common to many micxroparticles-based drug delivery systems [26, 31] . For the fi rst 4 hours, about (32.4±3.81)% BSA released from chitosan microspheres. At 240 h, about 70% BSA released for the microspheres. The release profiles may be closely related to the ways of the interactions between BSA molecules and chitosan microspheres. BSA molecules, when absorbed onto the surfaces of chitosan microspheres may released quickly, which constitutes the burst effects of the delivery systems, when the BSA molecules entered into the valleys of the coarse surfaces of the chitosan microspheres, the BSA molecules may acted as partial crosslinkers and released very slowly until the microspheres disintegrated, these may explain the sustained release profile of chitosan microspheres. In our another study [17] , the chitosan microspheres showed signifi cantly higher loading capacity for bovine coronavirus N protein than that for BSA. It is supposed that this may be caused by different isoelectric points of these proteins. As the pH 5.8 solution was used for adsorption of the different proteins by chitosan microspheres, that is, the larger distance between pH 5.8 and isoelectric points of the protein, the more electric charges carried by the protein molecules, and thus the more attrctive interactions between chitosan microspheres and protein molecules. Ma and Liu [21] prepared the BSA-loaded chitosan microspheres by ionotropic gelation using TPP and ethanol as solvents under a high voltage, the microspheres developed from different concentrations of coagulation solution showed pH-dependent release profi les.

The uniform-sized chitosan microspheres with size less than 10 μm can be prepared under a certain amount of crosslinkers. The chitosan microspheres containing BSA by absorptive method changed the characteristics of the blank chitosan microspheres as evidenced by FTIR and TG analysis. The obvious sustained process of the chitosan microspheres to BSA can be viewed.

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