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Preparation of chitosan microspheres by ionotropic gelation under a high voltage electrostatic field for protein delivery
Colloids and Surfaces B: Biointerfaces 75 (2010) 448–453
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Preparation of chitosan microspheres by ionotropic gelation under a high voltage electrostatic field for protein delivery Lihua Ma, Changsheng Liu ∗ Key Laboratory for Ultrafine Materials of Ministry of Education, Shanghai Key Laboratory of Advanced Polymeric Materials, and Engineering Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 12 January 2009 Received in revised form 16 September 2009 Accepted 16 September 2009 Available online 23 September 2009 Keywords: Chitosan Microspheres Protein Ionotropic gelation Encapsulation efficiency Release
a b s t r a c t Biodegradable microspheres have been widely used in drug/protein delivery system. In this paper, a modified ionotropic gelation method combined with a high voltage electrostatic field was developed to prepare protein-loaded chitosan microspheres. Bovine serum albumin (BSA) was chosen as a model protein. The preparation process and major parameters were discussed and optimized. The morphology, particle size, encapsulation efficiency and in vitro release behavior of the prepared microspheres were investigated. The results revealed that the microspheres exhibited good sphericity and dispersity when the mixture of sodium tripolyphosphate (TPP) and ethanol was applied as coagulation solution. Higher encapsulation efficiency (>90%) was achieved for the weight ratio of BSA to chitosan below 5%. 35% of BSA was released from the microspheres cured in 3% coagulation solution, and more than 50% of BSA was released from the microspheres cured in 1% coagulation solution at pH 8.8. However, only 15% of BSA was released from the microspheres cured in 1% coagulation solution at pH 4. The results suggested that ionotropic gelation method combined with a high voltage electrostatic field will be an effective method for fabricating chitosan microspheres for sustained delivery of protein. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Nowadays many investigations have been published on the delivery systems for protein and peptide drugs [1–4]. Among them, biodegradable microspheres have attracted great attention because they can protect proteins from degradation, be ingested or injected, or be tailored for desired release profiles, and in some cases even provide organ-targeted release [1]. Due to its biocompatibility, noninflammatory property, nontoxicity and biodegradability, chitosan, a naturally occurring polysaccharide composed of 2-amino-2deoxy-d-glucose and 2-acetamido-2-deoxy-d-glucose units linked with -(1 → 4) bonds, has been widely used in drug/protein release systems, such as antibiotics, proteins, and vaccines in the form of microspheres [5–9]. Various methods, including emulsification-solvent evaporation, emulsification–coacervation, phase separation–coacervation, ionotropic gelation and spray drying, have been developed for the fabrication of polymer microspheres [10–14]. Among them, emulsification method, due to the mild conditions such as ambient temperature, and preservation of the activity of core material, has been widely used in the past few years [10,11]. However, the repro-
∗ Corresponding author. Tel.: +86 21 64251308; fax: +86 21 64251358. E-mail address: [email protected] (C. Liu). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.09.018
ducibility of this method was poor from batch to batch and the particle size of the microspheres varied in a wide range, which in turn influenced the release behavior of drugs. Moreover, rudimental organic solvents and de-oiling processes in emulsification method decreased the bioavailability of drugs and impaired the biocompatibility of the microspheres. To overcome these drawbacks, ionotropic gelation method has been proposed [15,16]. For example, Kuo co-workers have prepared a series of multi- and single-walled drug-contained chitosan microspheres by treating micro-droplets of chitosan with mixing solutions of different ratios of TPP/NaOH under high voltage electrostatic field [15]. Unfortunately, proteins with high molecular weight, such as albumin could not be entrapped in the microspheres easily by such method. And the albumin-loaded chitosan microspheres became irregular in shape and fragile, with obvious burst release of albumin after initial incubation period. To resolve the problems, a modified ionotropic gelation method combined with a high voltage electrostatic field was developed for encapsulating large proteins into chitosan microspheres in this study. BSA, which has been employed in a number of investigations concerning sustained release [17–19], adsorption and interaction with various materials [20,21], was chosen as a model protein. To tailor the morphology and dispersity, the mixed solution of TPP and ethanol was used as coagulation solution. The morphology and particle size of the microspheres were examined. In addition,
L. Ma, C. Liu / Colloids and Surfaces B: Biointerfaces 75 (2010) 448–453
the effects of major preparation parameters on the encapsulation efficiency and in vitro release behaviors of BSA-loaded chitosan microspheres in phosphate buffered saline (PBS) were investigated. The main purpose of this study was to provide useful information on protein-loaded polymer microspheres fabricated by ionotropic gelation under a high voltage electrostatic field.
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where the weight of BSA in supernatant was quantified by Bradford protein assay. 1 ml supernatant from each sample was added to a centrifuge tube containing 4 ml Bradford reagent. Five minutes later, optical density (OD) was measured at 595 nm using UV spectrophotometer (UV-2550, Shimadzu, Japan). The weight of BSA in supernatant was determined from the established BSA standard curve.
2. Materials and methods 2.6. In vitro release profiles 2.1. Materials Chitosan (DD: 85%, Mv = 300,000) was purchased from Shanghai Kabo Industrial Trade Company. BSA (M = 68,000) was provided by Shanghai Yuanju Biotechnology Co. Ltd. TPP was obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). All other reagents were of reagent grade and were used without any further purification. 2.2. Microspheres fabrication BSA-loaded chitosan microspheres were fabricated via a modified ionotropic gelation method combined with a high voltage electrostatic field. Briefly, chitosan was dissolved in 2% (v/v) acetic acid aqueous solution, and then different amounts of BSA was respectively dissolved in chitosan solution. The resulted mixed solution was dripped into the coagulation solution containing aqueous TPP solution and ethanol through 6-gauge needle driven by a syringe pump (AJ5805, Shanghai Anjie Electronic Equipment Co. Ltd., China) under a high voltage electrostatic field. The needle was electrically connected with the positive electrode of a high voltage electrostatic generator, the negative electrode of which was electrically connected with an annular stainless steel plate fixed over the coagulation solution. The potential between the needle and the stainless steel plate was kept at 3.5 kV, and the pumping rate was 50 ml/h. The formed microspheres were collected by centrifugation, and the supernatant was kept for measuring the encapsulation efficiency. The collected microspheres were washed three times with deionized water, and then freeze-dried in a freeze dryer (FD-1, Beijing Boyikang Lab Instrument Co. Ltd., China). The dried chitosan microspheres were stored in a desiccator for use. Blank chitosan microspheres were fabricated by the same method except that no BSA was added.
Accurately weighted amounts of BSA-loaded chitosan microspheres were placed in 5 ml centrifuge tubes containing 1 ml PBS (pH 7.4), and incubated at 37 ◦ C in a bath shook at 100 rpm. At predetermined intervals, the supernatant fluid was collected and equivalent volume of fresh PBS was added to the centrifuge tube. The amount of BSA released was determined by Bradford protein assay. The amount of BSA released was normalized by the amount of BSA initially loaded into the microspheres. Chitosan microspheres were used as blank. 3. Results and discussion 3.1. Fabrication of microspheres In this study, chitosan microspheres were fabricated by ionotropic gelation method under a high voltage electrostatic field. The preparation scheme of the microspheres was shown in Fig. 1(a). Driven by the syringe pump, the original solution containing protein and chitosan flowed out of the hollow needle and was divided into liquid droplets by the high voltage electrostatic force. Liquid droplets reacted with coagulation solution, and then the microspheres were formed. The preparation process was simple and easy to operate. As for the ionotropic gelation method, coagulation solution is one of the important parameters. Glutaraldehyde (GA), ethylene
2.3. Morphology The morphology of the microspheres was observed under an inverted Microscope (TE2000-U, Nikon, Japan). The surface morphology of the microspheres was examined under a scanning electron microscope (JSM-6360LV, JEOL, Japan). Samples were adhered on a round brass stub and then sputter-coated with gold before morphology examination. 2.4. Particle size The mean diameter of wet microspheres was determined using the optical microscopy. The microscope eyepiece was filled with a micrometer by which the size of the microsphere could be determined. 2.5. Encapsulation efficiency Encapsulation efficiency (EE) was calculated as follows: EE (%) =
weight of starting BSA − weight of BSA in supernatant weight of starting BSA × 100
Fig. 1. Diagram of microspheres preparation. (a) Preparation process and (b) microspheres formation.
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Fig. 2. Scheme of microspheres cured in (a) TPP and (b) TPP and ethanol.
glycol diglycidyl ether (EGDE), and TPP [22–24] have been used as cross-linking agents for forming chitosan microspheres in previous studies. The results revealed that neither GA nor EGDE is the ideal cross-linking agent due to their physiological toxicity. In contrast, TPP is a non-toxic cross-linking agent, and multivalent negatively charged groups of TPP molecules can react with positively charged amino groups of chitosan, leading to the formation of chitosan gels. Mi et al. [24] also proposed that the ionic interaction between chitosan and TPP is pH-dependent. The reaction was briefly described in Fig. 1(b). When the pH of the coagulation solution was above 7, both OH− and P3 O5− 10 could diffuse into chitosan droplets to react with the protonated amino groups in chitosan. Coacervation phase-inversion accompanied by light ionic-cross-linking reaction occurred. When the pH was below 7, ionic-cross-linking reaction occurred because only P3 O5− 10 diffused into chitosan droplets to react with the protonated amino groups. The reaction between chitosan and TPP is mild under any pH condition, so TPP was chosen as ionic cross-linking agent in this study. To our knowledge, the specific gravity and the surface tension of the coagulation solution have great effect on the fabrication of the microspheres. Only when the specific gravity and the surface tension of the original solution are higher than those of the coagulation solution, chitosan droplets can immerse into the coagulation solution entirely and form microspheres therein. When TPP was used alone as coagulation solution, it was observed that most of chitosan droplets floated on the surface of the coagulation solution because the specific gravity of the original solution was close to that of the coagulation solution. And the ionic-cross-linking reaction between chitosan and TPP could not proceed completely (Fig. 2). As a result, BSA-loaded chitosan microspheres tended to adhere with each other. To resolve this problem, ethanol was added into the coagulation solution in this study. On the one hand, the specific gravity of ethanol is less than 1. On the other hand, the surface tension of ethanol (22.8 × 10−3 N/m) is less than that of water (72.8 × 10−3 N/m). Also, ethanol neither shows obvious toxicity nor reacts with TPP. Therefore, the addition of ethanol can decrease the specific gravity and the surface tension of the coagulation solution simultaneously. As expected, we found that the BSA-loaded chitosan droplets, after being ejected, quickly immersed into the coagulation solution, and microspheres with good sphericity and dispersity formed.
3.2. Characterization of microspheres It could be seen from the microscope that most of the formed microspheres had spherical shape and the surface of the microspheres was not smooth (Fig. 3). The amplified surface images of a single microsphere (Fig. 4) showed that the composition and the pH
Fig. 3. Photomicrographs of microspheres. (a) 100× and (b) 200×.
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Fig. 5. The effect of coagulation solution and chitosan concentration on the particle size of the microspheres. Each point represents the mean ± S.D. (n = 50).
of the coagulation solution have obvious effect on the morphology of chitosan microspheres. When TPP was used alone as coagulation solution, the surface of BSA-loaded chitosan microspheres seemed scraggly. And when the mixture of TPP and ethanol was used, a crimple structure was produced in spite of the loading of BSA. More crimples appeared on the surface of BSA-loaded chitosan microspheres when the coagulation solution was adjusted from basic solution to acidic solution. It is also noted that most crimples were observed on the surface of blank chitosan microspheres. During the freeze-drying process, plenty of water was removed from the microspheres and crimple structure was formed, which enlarged the superficial area of the microspheres and provided channels of protein from the microspheres. The particle size of the microspheres could be adjusted by varying chitosan concentration. Fig. 5 showed that the particle size in the range of 100–450 m could be obtained by adjusting the concentration of chitosan from 0.5 to 1.2 g/l. Also, the particle size of the microspheres cured in the mixture of TPP and ethanol was a little larger than that of the microspheres cured in TPP. Due to ethanol addition, the sedimentation rate of liquid droplets is greater than gelation rate. Liquid droplets got larger in the course of immersing into coagulation solution. So the particle size of the microspheres cured in TPP and ethanol was relatively large. The concentration and pH of the coagulation solution had no obvious effects on the particle size of the microspheres. The results indicated that the particle size was mainly regulated by the state of the original solution flowed out from the needle. 3.3. Encapsulation efficiency
Fig. 4. SEM photographs of (a) chitosan microsphere cured in TPP and ethanol at pH 4, (b) BSA-loaded chitosan microsphere cured in TPP and ethanol at pH 4, (c) BSA-loaded chitosan microsphere cured in TPP at pH 4, and (d) BSA-loaded chitosan microsphere cured in TPP and ethanol at pH 8.8.
To prevent the loss of precious protein and extend the duration and dosage of treatment, it is desirable to increase the encapsulation efficiency. In the present study, to achieve maximal the encapsulation efficiency, the main experimental conditions, such as the composition of the coagulation solution, the weight ratio of BSA to chitosan, and the pH of the coagulation solution were investigated. Fig. 6 showed that the incorporation of ethanol into coagulation solution slightly decreased the encapsulation efficiency. From Fig. 7, it could be seen that the encapsulation efficiency decreased with the increase of the weight ratio BSA to chitosan. Especially, the encapsulation efficiency was less than 40% when the weight ratio of BSA to chitosan reached 20%. As we know, with the increase of the weight ratio of BSA to chitosan, NH+ exposed on the surface of liquid droplets is decreased. 3
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Fig. 6. The effect of coagulation solution on the encapsulation efficiency. Each point represents the mean ± S.D. (n = 6).
Sites for ionic interactions between chitosan and TPP on the surface of liquid droplets are reduced, and thus the stability of the microspheres is decreased. Therefore, the tight gelatin structure cannot be formed, and BSA cannot be entrapped into the microspheres completely. Similarly, decreasing the concentration of the coagulation solution also resulted in lower encapsulation efficiency. To investigate the effect of pH of the coagulation solution, in this study, the pH value of the coagulation solution was modulated from 4 to 8.8, and the encapsulation efficiency of BSA-loaded chitosan microspheres was examined. As shown in Fig. 8, the pH of the coagulation solution indeed had obvious effect on the encapsulation efficiency, and the encapsulation efficiency increased with the decrease of the pH of the coagulation solution. It is believed that this is due to different gelatin structures formed in the coagulation solution under different pH values. To summary, the investigation on the encapsulation efficiency clearly demonstrated that BSA-loaded chitosan microspheres with higher encapsulation efficiency could be obtained by using ionotropic gelation method combined with a high voltage electrostatic field and keeping the weight ratio of BSA to chitosan below 5%.
Fig. 7. The effect of BSA amount on the encapsulation efficiency. Each point represents the mean ± S.D. (n = 6).
Fig. 8. The effect of pH of the coagulation solution on the encapsulation efficiency. Each point represents the mean ± S.D. (n = 6).
3.4. In vitro protein release Controlled release is another desirable characteristic for drug delivery systems. In this study, the release of BSA from BSA-loaded chitosan microspheres fabricated under various conditions was conducted in PBS at 37 ◦ C, and the release profiles were showed in Figs. 9 and 10. At the first glance, the weight percentage of BSA cumulative release indicated a sustained release over one week. There was an initial quick release followed in a second stage by a slow release. The quick release was caused by the release of BSA close to the surface of the microspheres. In addition, the release profiles were significantly affected by the concentration and pH of the coagulation solution. It was observed from Fig. 9 that the release rate was increased with the decrease of the concentration of the coagulation solution. 35% of BSA was released from chitosan microspheres cured in 3% coagulation solution, and more than 50% of BSA was released from chitosan microspheres cured in 1% coagulation solution at pH 8.8. In the case of the microspheres cured in 1% coagulation solution at pH 4, the release rate was very low, and only 15% of BSA was released after 7 days (Fig. 10). Generally, the release of protein from polymer microspheres is controlled by both protein
Fig. 9. BSA release profiles from chitosan microspheres fabricated with different concentrations of the coagulation solution. Each point represents the mean ± S.D. (n = 4).
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release of BSA could be regulated by the concentration and pH of the coagulation solution. The microspheres offered high BSA encapsulation efficiency and sustained release of proteins. The results indicated that the ionotropic gelation method combined with a high voltage electrostatic field will be an effective tool to prepare microspheres, the latter offering potential biomedical applications such as embolization agent of vein, buccal mucosa delivery system, and tissue engineering. Acknowledgements The authors would like to acknowledge funding supported by Major Program of National Natural Science Foundation of China (No. 50732002), National Key Technology R&D Program (No. 2006BAI16B02), Shanghai Leading Academic Discipline Project (No. B502), and Shanghai Key Laboratory Project (No. 08DZ2230500). References
Fig. 10. BSA release profiles from chitosan microspheres fabricated with different pHs of the coagulation solution. Each point represents the mean ± S.D. (n = 4).
diffusion and polymer degradation. In this study, the degradation of chitosan microspheres was not observed during the experiments, which has been demonstrated in previous studies [25,26]. Therefore, the release of protein from chitosan microspheres was mainly controlled by protein diffusion. As mentioned above, when the concentration of the coagulation solution was increased from 1% to 3% or the pH value of the coagulation solution was adjusted from 8.8 to 4, the cross-linking sites increased. As a result, chitosan microspheres with tighter structure were formed and the diffusion rate of BSA decreased correspondingly. In other words, the release behavior of BSA from chitosan microspheres fabricated via ionotropic gelation method combined with a high voltage electrostatic field could be controlled by adjusting the concentration and pH of the coagulation solution. Further investigations are on the way for investigating the activity and the long-term release behavior of protein.
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
4. Conclusion In this work, chitosan microspheres containing protein with high molecular weight were prepared via a modified ionotropic gelation method under a high voltage electrostatic field. The mixture of TPP and ethanol was chosen as coagulation solution. The microspheres as prepared exhibited good sphericity and dispersity. The particle size of the microspheres could be adjusted by chitosan concentration and the encapsulation efficiency and in vitro
[18] [19] [20] [21] [22] [23] [24] [25] [26]
V.R. Sinha, A. Trehan, J. Control. Release 90 (2003) 261–280. P.T. Vladimir, N.L. Anatoly, Drug Discov. Today 8 (2003) 259–266. M.A. Welte, Trends Cell Biol. 17 (2007) 363–369. K.T. Joerg, M.G. Achim, Adv. Drug Deliv. Rev. 59 (2007) 274–291. V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan, Int. J. Pharm. 274 (2004) 1–33. ´ n-López, Int. J. Pharm. 312 (2006) 166– ˜␣ A.K. Anal, W.F. Stevens, C. Remun 173. S. Shanmuganathan, N. Shanumugasundaram, N. Adhirajan, T.S.R. Lakshmi, M. Babu, Carbohydr. Polym. 73 (2008) 201–211. W. Wei, L. Yuan, G. Hu, L.Y. Wang, H. Wu, X. Hu, Z.G. Su, G.H. Ma, Adv. Mater. 20 (2008) 2292–2296. J.Y. Tian, J. Yu, X.Q. Sun, Vet. Immunol. Immunopathol. 126 (2008) 220–229. S.E. Kim, J.H. Park, Y.W. Cho, H. Chung, S.Y. Jeong, E.B. Lee, I.C. Kwon, J. Control. Release 91 (2003) 365–374. J.E. Lee, K.E. Kim, I.C. Kwon, H.J. Ahn, S.H. Lee, H. Cho, H.J. Kim, S.C. Seong, M.C. Lee, Biomaterials 25 (2004) 4163–4173. P. Perugini, I. Genta, F. Pavanetto, B. Conti, S. Scalia, A. Baruffini, Int. J. Pharm. 196 (2000) 51–61. J. Berge, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Eur. J. Pharm. Biopharm. 57 (2004) 19–34. P. He, S.S. Davis, L. Illum, Int. J. Pharm. 187 (1999) 53–65. S.J. Chang, G.C. Niu, S.M. Kuo, S.F. Chen, J. Biomed. Mater. Res. 81 (2006) 554–566. S.J. Chang, S.M. Kuo, I. Manousakas, G.C. Niu, J.P. Chen, Acta Biomater. 5 (2009) 101–114. K. Maldenovska, E.F. Kumbaradzi, G.M. Dodov, L. Makraduli, K. Goracinova, Int. J. Pharm. 242 (2002) 247–249. J. Xie, C. Wang, Biotech. Bioeng. 97 (2007) 1278–1290. C.Y. Yu, B.C. Yin, W. Zhang, S.X. Cheng, X.Z. Zhang, R.X. Zhuo, Colloids Surf. B: Biointerf. 68 (2009) 245–249. Y.H. Miao, L.E. Helseth, Colloids Surf. B: Biointerf. 66 (2008) 299–303. E.M. Pinto, D.M. Soares, C.M.A. Brett, Electrochim. Acta 53 (2008) 7460–7466. A. Jayakrishnan, S.R. Jameela, Biomaterials 17 (1996) 471–484. X.F. Zeng, E. Ruckenstein, J. Membr. Sci. 148 (1998) 195–205. F.L. Mi, S.S. Shyu, S.T. Lee, T.B. Wong, J. Polym. Sci. B: Polym. Phys. 37 (1999) 1551–1564. K. Tomihata, Y. Ikada, Biomaterials 18 (1997) 567–575. D.W. Ren, H.F. Y, W. Wang, X.J. Ma, Carbohydr. Res. 340 (2005) 2403–2410.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Preparation of chitosan microspheres by ionotropic gelation under a high voltage electrostatic field for protein delivery Lihua Ma, Changsheng Liu ∗ Key Laboratory for Ultrafine Materials of Ministry of Education, Shanghai Key Laboratory of Advanced Polymeric Materials, and Engineering Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 12 January 2009 Received in revised form 16 September 2009 Accepted 16 September 2009 Available online 23 September 2009 Keywords: Chitosan Microspheres Protein Ionotropic gelation Encapsulation efficiency Release
a b s t r a c t Biodegradable microspheres have been widely used in drug/protein delivery system. In this paper, a modified ionotropic gelation method combined with a high voltage electrostatic field was developed to prepare protein-loaded chitosan microspheres. Bovine serum albumin (BSA) was chosen as a model protein. The preparation process and major parameters were discussed and optimized. The morphology, particle size, encapsulation efficiency and in vitro release behavior of the prepared microspheres were investigated. The results revealed that the microspheres exhibited good sphericity and dispersity when the mixture of sodium tripolyphosphate (TPP) and ethanol was applied as coagulation solution. Higher encapsulation efficiency (>90%) was achieved for the weight ratio of BSA to chitosan below 5%. 35% of BSA was released from the microspheres cured in 3% coagulation solution, and more than 50% of BSA was released from the microspheres cured in 1% coagulation solution at pH 8.8. However, only 15% of BSA was released from the microspheres cured in 1% coagulation solution at pH 4. The results suggested that ionotropic gelation method combined with a high voltage electrostatic field will be an effective method for fabricating chitosan microspheres for sustained delivery of protein. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Nowadays many investigations have been published on the delivery systems for protein and peptide drugs [1–4]. Among them, biodegradable microspheres have attracted great attention because they can protect proteins from degradation, be ingested or injected, or be tailored for desired release profiles, and in some cases even provide organ-targeted release [1]. Due to its biocompatibility, noninflammatory property, nontoxicity and biodegradability, chitosan, a naturally occurring polysaccharide composed of 2-amino-2deoxy-d-glucose and 2-acetamido-2-deoxy-d-glucose units linked with -(1 → 4) bonds, has been widely used in drug/protein release systems, such as antibiotics, proteins, and vaccines in the form of microspheres [5–9]. Various methods, including emulsification-solvent evaporation, emulsification–coacervation, phase separation–coacervation, ionotropic gelation and spray drying, have been developed for the fabrication of polymer microspheres [10–14]. Among them, emulsification method, due to the mild conditions such as ambient temperature, and preservation of the activity of core material, has been widely used in the past few years [10,11]. However, the repro-
∗ Corresponding author. Tel.: +86 21 64251308; fax: +86 21 64251358. E-mail address: [email protected] (C. Liu). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.09.018
ducibility of this method was poor from batch to batch and the particle size of the microspheres varied in a wide range, which in turn influenced the release behavior of drugs. Moreover, rudimental organic solvents and de-oiling processes in emulsification method decreased the bioavailability of drugs and impaired the biocompatibility of the microspheres. To overcome these drawbacks, ionotropic gelation method has been proposed [15,16]. For example, Kuo co-workers have prepared a series of multi- and single-walled drug-contained chitosan microspheres by treating micro-droplets of chitosan with mixing solutions of different ratios of TPP/NaOH under high voltage electrostatic field [15]. Unfortunately, proteins with high molecular weight, such as albumin could not be entrapped in the microspheres easily by such method. And the albumin-loaded chitosan microspheres became irregular in shape and fragile, with obvious burst release of albumin after initial incubation period. To resolve the problems, a modified ionotropic gelation method combined with a high voltage electrostatic field was developed for encapsulating large proteins into chitosan microspheres in this study. BSA, which has been employed in a number of investigations concerning sustained release [17–19], adsorption and interaction with various materials [20,21], was chosen as a model protein. To tailor the morphology and dispersity, the mixed solution of TPP and ethanol was used as coagulation solution. The morphology and particle size of the microspheres were examined. In addition,
L. Ma, C. Liu / Colloids and Surfaces B: Biointerfaces 75 (2010) 448–453
the effects of major preparation parameters on the encapsulation efficiency and in vitro release behaviors of BSA-loaded chitosan microspheres in phosphate buffered saline (PBS) were investigated. The main purpose of this study was to provide useful information on protein-loaded polymer microspheres fabricated by ionotropic gelation under a high voltage electrostatic field.
449
where the weight of BSA in supernatant was quantified by Bradford protein assay. 1 ml supernatant from each sample was added to a centrifuge tube containing 4 ml Bradford reagent. Five minutes later, optical density (OD) was measured at 595 nm using UV spectrophotometer (UV-2550, Shimadzu, Japan). The weight of BSA in supernatant was determined from the established BSA standard curve.
2. Materials and methods 2.6. In vitro release profiles 2.1. Materials Chitosan (DD: 85%, Mv = 300,000) was purchased from Shanghai Kabo Industrial Trade Company. BSA (M = 68,000) was provided by Shanghai Yuanju Biotechnology Co. Ltd. TPP was obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). All other reagents were of reagent grade and were used without any further purification. 2.2. Microspheres fabrication BSA-loaded chitosan microspheres were fabricated via a modified ionotropic gelation method combined with a high voltage electrostatic field. Briefly, chitosan was dissolved in 2% (v/v) acetic acid aqueous solution, and then different amounts of BSA was respectively dissolved in chitosan solution. The resulted mixed solution was dripped into the coagulation solution containing aqueous TPP solution and ethanol through 6-gauge needle driven by a syringe pump (AJ5805, Shanghai Anjie Electronic Equipment Co. Ltd., China) under a high voltage electrostatic field. The needle was electrically connected with the positive electrode of a high voltage electrostatic generator, the negative electrode of which was electrically connected with an annular stainless steel plate fixed over the coagulation solution. The potential between the needle and the stainless steel plate was kept at 3.5 kV, and the pumping rate was 50 ml/h. The formed microspheres were collected by centrifugation, and the supernatant was kept for measuring the encapsulation efficiency. The collected microspheres were washed three times with deionized water, and then freeze-dried in a freeze dryer (FD-1, Beijing Boyikang Lab Instrument Co. Ltd., China). The dried chitosan microspheres were stored in a desiccator for use. Blank chitosan microspheres were fabricated by the same method except that no BSA was added.
Accurately weighted amounts of BSA-loaded chitosan microspheres were placed in 5 ml centrifuge tubes containing 1 ml PBS (pH 7.4), and incubated at 37 ◦ C in a bath shook at 100 rpm. At predetermined intervals, the supernatant fluid was collected and equivalent volume of fresh PBS was added to the centrifuge tube. The amount of BSA released was determined by Bradford protein assay. The amount of BSA released was normalized by the amount of BSA initially loaded into the microspheres. Chitosan microspheres were used as blank. 3. Results and discussion 3.1. Fabrication of microspheres In this study, chitosan microspheres were fabricated by ionotropic gelation method under a high voltage electrostatic field. The preparation scheme of the microspheres was shown in Fig. 1(a). Driven by the syringe pump, the original solution containing protein and chitosan flowed out of the hollow needle and was divided into liquid droplets by the high voltage electrostatic force. Liquid droplets reacted with coagulation solution, and then the microspheres were formed. The preparation process was simple and easy to operate. As for the ionotropic gelation method, coagulation solution is one of the important parameters. Glutaraldehyde (GA), ethylene
2.3. Morphology The morphology of the microspheres was observed under an inverted Microscope (TE2000-U, Nikon, Japan). The surface morphology of the microspheres was examined under a scanning electron microscope (JSM-6360LV, JEOL, Japan). Samples were adhered on a round brass stub and then sputter-coated with gold before morphology examination. 2.4. Particle size The mean diameter of wet microspheres was determined using the optical microscopy. The microscope eyepiece was filled with a micrometer by which the size of the microsphere could be determined. 2.5. Encapsulation efficiency Encapsulation efficiency (EE) was calculated as follows: EE (%) =
weight of starting BSA − weight of BSA in supernatant weight of starting BSA × 100
Fig. 1. Diagram of microspheres preparation. (a) Preparation process and (b) microspheres formation.
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Fig. 2. Scheme of microspheres cured in (a) TPP and (b) TPP and ethanol.
glycol diglycidyl ether (EGDE), and TPP [22–24] have been used as cross-linking agents for forming chitosan microspheres in previous studies. The results revealed that neither GA nor EGDE is the ideal cross-linking agent due to their physiological toxicity. In contrast, TPP is a non-toxic cross-linking agent, and multivalent negatively charged groups of TPP molecules can react with positively charged amino groups of chitosan, leading to the formation of chitosan gels. Mi et al. [24] also proposed that the ionic interaction between chitosan and TPP is pH-dependent. The reaction was briefly described in Fig. 1(b). When the pH of the coagulation solution was above 7, both OH− and P3 O5− 10 could diffuse into chitosan droplets to react with the protonated amino groups in chitosan. Coacervation phase-inversion accompanied by light ionic-cross-linking reaction occurred. When the pH was below 7, ionic-cross-linking reaction occurred because only P3 O5− 10 diffused into chitosan droplets to react with the protonated amino groups. The reaction between chitosan and TPP is mild under any pH condition, so TPP was chosen as ionic cross-linking agent in this study. To our knowledge, the specific gravity and the surface tension of the coagulation solution have great effect on the fabrication of the microspheres. Only when the specific gravity and the surface tension of the original solution are higher than those of the coagulation solution, chitosan droplets can immerse into the coagulation solution entirely and form microspheres therein. When TPP was used alone as coagulation solution, it was observed that most of chitosan droplets floated on the surface of the coagulation solution because the specific gravity of the original solution was close to that of the coagulation solution. And the ionic-cross-linking reaction between chitosan and TPP could not proceed completely (Fig. 2). As a result, BSA-loaded chitosan microspheres tended to adhere with each other. To resolve this problem, ethanol was added into the coagulation solution in this study. On the one hand, the specific gravity of ethanol is less than 1. On the other hand, the surface tension of ethanol (22.8 × 10−3 N/m) is less than that of water (72.8 × 10−3 N/m). Also, ethanol neither shows obvious toxicity nor reacts with TPP. Therefore, the addition of ethanol can decrease the specific gravity and the surface tension of the coagulation solution simultaneously. As expected, we found that the BSA-loaded chitosan droplets, after being ejected, quickly immersed into the coagulation solution, and microspheres with good sphericity and dispersity formed.
3.2. Characterization of microspheres It could be seen from the microscope that most of the formed microspheres had spherical shape and the surface of the microspheres was not smooth (Fig. 3). The amplified surface images of a single microsphere (Fig. 4) showed that the composition and the pH
Fig. 3. Photomicrographs of microspheres. (a) 100× and (b) 200×.
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Fig. 5. The effect of coagulation solution and chitosan concentration on the particle size of the microspheres. Each point represents the mean ± S.D. (n = 50).
of the coagulation solution have obvious effect on the morphology of chitosan microspheres. When TPP was used alone as coagulation solution, the surface of BSA-loaded chitosan microspheres seemed scraggly. And when the mixture of TPP and ethanol was used, a crimple structure was produced in spite of the loading of BSA. More crimples appeared on the surface of BSA-loaded chitosan microspheres when the coagulation solution was adjusted from basic solution to acidic solution. It is also noted that most crimples were observed on the surface of blank chitosan microspheres. During the freeze-drying process, plenty of water was removed from the microspheres and crimple structure was formed, which enlarged the superficial area of the microspheres and provided channels of protein from the microspheres. The particle size of the microspheres could be adjusted by varying chitosan concentration. Fig. 5 showed that the particle size in the range of 100–450 m could be obtained by adjusting the concentration of chitosan from 0.5 to 1.2 g/l. Also, the particle size of the microspheres cured in the mixture of TPP and ethanol was a little larger than that of the microspheres cured in TPP. Due to ethanol addition, the sedimentation rate of liquid droplets is greater than gelation rate. Liquid droplets got larger in the course of immersing into coagulation solution. So the particle size of the microspheres cured in TPP and ethanol was relatively large. The concentration and pH of the coagulation solution had no obvious effects on the particle size of the microspheres. The results indicated that the particle size was mainly regulated by the state of the original solution flowed out from the needle. 3.3. Encapsulation efficiency
Fig. 4. SEM photographs of (a) chitosan microsphere cured in TPP and ethanol at pH 4, (b) BSA-loaded chitosan microsphere cured in TPP and ethanol at pH 4, (c) BSA-loaded chitosan microsphere cured in TPP at pH 4, and (d) BSA-loaded chitosan microsphere cured in TPP and ethanol at pH 8.8.
To prevent the loss of precious protein and extend the duration and dosage of treatment, it is desirable to increase the encapsulation efficiency. In the present study, to achieve maximal the encapsulation efficiency, the main experimental conditions, such as the composition of the coagulation solution, the weight ratio of BSA to chitosan, and the pH of the coagulation solution were investigated. Fig. 6 showed that the incorporation of ethanol into coagulation solution slightly decreased the encapsulation efficiency. From Fig. 7, it could be seen that the encapsulation efficiency decreased with the increase of the weight ratio BSA to chitosan. Especially, the encapsulation efficiency was less than 40% when the weight ratio of BSA to chitosan reached 20%. As we know, with the increase of the weight ratio of BSA to chitosan, NH+ exposed on the surface of liquid droplets is decreased. 3
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Fig. 6. The effect of coagulation solution on the encapsulation efficiency. Each point represents the mean ± S.D. (n = 6).
Sites for ionic interactions between chitosan and TPP on the surface of liquid droplets are reduced, and thus the stability of the microspheres is decreased. Therefore, the tight gelatin structure cannot be formed, and BSA cannot be entrapped into the microspheres completely. Similarly, decreasing the concentration of the coagulation solution also resulted in lower encapsulation efficiency. To investigate the effect of pH of the coagulation solution, in this study, the pH value of the coagulation solution was modulated from 4 to 8.8, and the encapsulation efficiency of BSA-loaded chitosan microspheres was examined. As shown in Fig. 8, the pH of the coagulation solution indeed had obvious effect on the encapsulation efficiency, and the encapsulation efficiency increased with the decrease of the pH of the coagulation solution. It is believed that this is due to different gelatin structures formed in the coagulation solution under different pH values. To summary, the investigation on the encapsulation efficiency clearly demonstrated that BSA-loaded chitosan microspheres with higher encapsulation efficiency could be obtained by using ionotropic gelation method combined with a high voltage electrostatic field and keeping the weight ratio of BSA to chitosan below 5%.
Fig. 7. The effect of BSA amount on the encapsulation efficiency. Each point represents the mean ± S.D. (n = 6).
Fig. 8. The effect of pH of the coagulation solution on the encapsulation efficiency. Each point represents the mean ± S.D. (n = 6).
3.4. In vitro protein release Controlled release is another desirable characteristic for drug delivery systems. In this study, the release of BSA from BSA-loaded chitosan microspheres fabricated under various conditions was conducted in PBS at 37 ◦ C, and the release profiles were showed in Figs. 9 and 10. At the first glance, the weight percentage of BSA cumulative release indicated a sustained release over one week. There was an initial quick release followed in a second stage by a slow release. The quick release was caused by the release of BSA close to the surface of the microspheres. In addition, the release profiles were significantly affected by the concentration and pH of the coagulation solution. It was observed from Fig. 9 that the release rate was increased with the decrease of the concentration of the coagulation solution. 35% of BSA was released from chitosan microspheres cured in 3% coagulation solution, and more than 50% of BSA was released from chitosan microspheres cured in 1% coagulation solution at pH 8.8. In the case of the microspheres cured in 1% coagulation solution at pH 4, the release rate was very low, and only 15% of BSA was released after 7 days (Fig. 10). Generally, the release of protein from polymer microspheres is controlled by both protein
Fig. 9. BSA release profiles from chitosan microspheres fabricated with different concentrations of the coagulation solution. Each point represents the mean ± S.D. (n = 4).
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release of BSA could be regulated by the concentration and pH of the coagulation solution. The microspheres offered high BSA encapsulation efficiency and sustained release of proteins. The results indicated that the ionotropic gelation method combined with a high voltage electrostatic field will be an effective tool to prepare microspheres, the latter offering potential biomedical applications such as embolization agent of vein, buccal mucosa delivery system, and tissue engineering. Acknowledgements The authors would like to acknowledge funding supported by Major Program of National Natural Science Foundation of China (No. 50732002), National Key Technology R&D Program (No. 2006BAI16B02), Shanghai Leading Academic Discipline Project (No. B502), and Shanghai Key Laboratory Project (No. 08DZ2230500). References
Fig. 10. BSA release profiles from chitosan microspheres fabricated with different pHs of the coagulation solution. Each point represents the mean ± S.D. (n = 4).
diffusion and polymer degradation. In this study, the degradation of chitosan microspheres was not observed during the experiments, which has been demonstrated in previous studies [25,26]. Therefore, the release of protein from chitosan microspheres was mainly controlled by protein diffusion. As mentioned above, when the concentration of the coagulation solution was increased from 1% to 3% or the pH value of the coagulation solution was adjusted from 8.8 to 4, the cross-linking sites increased. As a result, chitosan microspheres with tighter structure were formed and the diffusion rate of BSA decreased correspondingly. In other words, the release behavior of BSA from chitosan microspheres fabricated via ionotropic gelation method combined with a high voltage electrostatic field could be controlled by adjusting the concentration and pH of the coagulation solution. Further investigations are on the way for investigating the activity and the long-term release behavior of protein.
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
4. Conclusion In this work, chitosan microspheres containing protein with high molecular weight were prepared via a modified ionotropic gelation method under a high voltage electrostatic field. The mixture of TPP and ethanol was chosen as coagulation solution. The microspheres as prepared exhibited good sphericity and dispersity. The particle size of the microspheres could be adjusted by chitosan concentration and the encapsulation efficiency and in vitro
[18] [19] [20] [21] [22] [23] [24] [25] [26]
V.R. Sinha, A. Trehan, J. Control. Release 90 (2003) 261–280. P.T. Vladimir, N.L. Anatoly, Drug Discov. Today 8 (2003) 259–266. M.A. Welte, Trends Cell Biol. 17 (2007) 363–369. K.T. Joerg, M.G. Achim, Adv. Drug Deliv. Rev. 59 (2007) 274–291. V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan, Int. J. Pharm. 274 (2004) 1–33. ´ n-López, Int. J. Pharm. 312 (2006) 166– ˜␣ A.K. Anal, W.F. Stevens, C. Remun 173. S. Shanmuganathan, N. Shanumugasundaram, N. Adhirajan, T.S.R. Lakshmi, M. Babu, Carbohydr. Polym. 73 (2008) 201–211. W. Wei, L. Yuan, G. Hu, L.Y. Wang, H. Wu, X. Hu, Z.G. Su, G.H. Ma, Adv. Mater. 20 (2008) 2292–2296. J.Y. Tian, J. Yu, X.Q. Sun, Vet. Immunol. Immunopathol. 126 (2008) 220–229. S.E. Kim, J.H. Park, Y.W. Cho, H. Chung, S.Y. Jeong, E.B. Lee, I.C. Kwon, J. Control. Release 91 (2003) 365–374. J.E. Lee, K.E. Kim, I.C. Kwon, H.J. Ahn, S.H. Lee, H. Cho, H.J. Kim, S.C. Seong, M.C. Lee, Biomaterials 25 (2004) 4163–4173. P. Perugini, I. Genta, F. Pavanetto, B. Conti, S. Scalia, A. Baruffini, Int. J. Pharm. 196 (2000) 51–61. J. Berge, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Eur. J. Pharm. Biopharm. 57 (2004) 19–34. P. He, S.S. Davis, L. Illum, Int. J. Pharm. 187 (1999) 53–65. S.J. Chang, G.C. Niu, S.M. Kuo, S.F. Chen, J. Biomed. Mater. Res. 81 (2006) 554–566. S.J. Chang, S.M. Kuo, I. Manousakas, G.C. Niu, J.P. Chen, Acta Biomater. 5 (2009) 101–114. K. Maldenovska, E.F. Kumbaradzi, G.M. Dodov, L. Makraduli, K. Goracinova, Int. J. Pharm. 242 (2002) 247–249. J. Xie, C. Wang, Biotech. Bioeng. 97 (2007) 1278–1290. C.Y. Yu, B.C. Yin, W. Zhang, S.X. Cheng, X.Z. Zhang, R.X. Zhuo, Colloids Surf. B: Biointerf. 68 (2009) 245–249. Y.H. Miao, L.E. Helseth, Colloids Surf. B: Biointerf. 66 (2008) 299–303. E.M. Pinto, D.M. Soares, C.M.A. Brett, Electrochim. Acta 53 (2008) 7460–7466. A. Jayakrishnan, S.R. Jameela, Biomaterials 17 (1996) 471–484. X.F. Zeng, E. Ruckenstein, J. Membr. Sci. 148 (1998) 195–205. F.L. Mi, S.S. Shyu, S.T. Lee, T.B. Wong, J. Polym. Sci. B: Polym. Phys. 37 (1999) 1551–1564. K. Tomihata, Y. Ikada, Biomaterials 18 (1997) 567–575. D.W. Ren, H.F. Y, W. Wang, X.J. Ma, Carbohydr. Res. 340 (2005) 2403–2410.