ARTICLE pubs.acs.org/IECR
Size Control of Chitosan Capsules Containing Insulin for Oral Drug Delivery via a Combined Process of Ionic Gelation with Electrohydrodynamic Atomization Sang-Yoon Kim,† Hyunah Lee,‡ Sungyeon Cho,‡ Ji-Woon Park,† Jiyong Park,‡ and Jungho Hwang†,* † ‡
Department of Mechanical Engineering, Yonsei University, Shinchon-dong, 120-749, Seoul, Korea Department of Biotechnology, Yonsei University, Shinchon-dong, 120-749, Seoul, Korea ABSTRACT: Recently, the development of new insulin delivery strategies, such as oral drug delivery, has sparked the interest of many researchers. In this paper, microsized chitosan capsules containing insulin were produced via a combined process of ionic gelation with electrohydrodynamic atomization (EHDA). Produced airborne chitosaninsulin droplets were reacted with phytic acid to induce the binding between chitosan and phytic acid, resulting in chitosan capsules containing insulin. As the nozzle size decreased, the size and uniformity of the primary airborne droplets decreased and enhanced, respectively. The size of primary airborne droplets affected the uniformity of the chitosan capsules in size. Using a nozzle of 320 μm in diameter, the mean diameter of the capsules was 232 μm, which is much smaller than that of capsules formed using only the ionic gelation method. The insulin encapsulation efficiency was nearly independent of nozzle size and was above 92%. Through in vitro tests, when a nozzle smaller than or equal to about 700 μm was used, the sustained insulin release occurred in the artificial intestinal juice although the smaller capsules exhibited lower resistance to acidic conditions.
1. INTRODUCTION Patients with diabetes mellitus require insulin therapy by injection. The treatment by injection involves one or more daily doses of intermediate- or long-acting insulin injections, as well as an injection before each meal.1 The approach is satisfactorily efficient; however, it brings distress and inconvenience to patients as well as induces unstable curative effects and side effects. Recently, the development of new insulin delivery strategies, such as oral delivery, has sparked interest of many researchers. Insulin can be conveniently delivered through oral administration. However, the method through oral administration possesses several limitations, which are low bioavailability because of degradation in the stomach, inactivation and digestion by proteolytic enzymes in the luminal cavity, and poor permeability across the intestinal epithelium.2 One attempt to overcome these problems is the use of an encapsulation process to provide protection for the insulin. Oral insulin delivery strategies are based on microcapsule carriers. Microcapsule carriers are small in size, allowing them to pass the intestinal mucosa, thus increasing the effectiveness and providing a more uniform distribution in the gastrointestinal tract. Additionally, their large specific area favors their capacity as a loading drug.3,4 Generally, chitosan has been used as an encapsulating agent for the oral delivery of diabetic medications. Chitosan reportedly interacts with insulin and enhances intestinal permeation and the stability of insulin via an oral route due to its good biocompability, bioadhesiveness, and biodegradability.5 Several methods have been employed for the synthesis of chitosan capsules including solvent emulsification/solvent evaporation,68 coacervation/precipitation,912 and spray-drying.1315 The solution must be stirred for extended periods of time when employing the aforementioned methods since chemical cross-linking agents potentially induce undesirable toxic effects if left unremoved.16 Complete r 2011 American Chemical Society
removal of unreacted cross-linking agents is often difficult in the aforementioned processes, which in turn has limited the utilization of cross-linked chitosan microcapsules in the pharmaceutical field.17 Further, the use of high speed stirring during the synthesis process interferes with the activity of the bioactive agents and can affect the mechanical properties of the capsules.18 The ionic gelation method has been used to prepare the chitosan capsules without chemical cross-linking agents. The ionic gelation method uses complexation between chitosan and oppositely charged macromolecules, and is commonly used to prepare the capsules.1922 Chitosan molecules in aqueous solutions adopt extended conformation with a more flexible chain because of the electrostatic charge repulsion between the chains. Chitosan has primary amino groups that have pKa values of about 6.3. An acid dissociation constant (Ka) is a quantitative measure of the strength of an acid in solution. Because of many orders of magnitude spanned by Ka values, a logarithmic measure of the acid dissociation constant (pKa) is more commonly used. At pH values below the pKa, most of the amino groups are protonated.23 In an acidic solution, the NH2 of chitosan molecule is protonized to be NH3+, interacting with an anion cross-linking agent as phytic acid (PA) by ionic interaction to form the capsules.24 PA is a well-known naturally occurring acid and nontoxic, biocompatible material. Structurally, PA has six acid groups attached symmetrically to a cyclohexamehexol ring, and each of them can dissociate and donate two protons, Among the 12 proton dissociation site, six are strongly acidic with an approximate pKa value Received: April 28, 2011 Accepted: October 31, 2011 Revised: September 13, 2011 Published: October 31, 2011 13762
dx.doi.org/10.1021/ie200915x | Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research
ARTICLE
of 1.5, three are weakley acidic with pKa values between 5.7 and 7.6, and the remaining three are very weakly acidic with pKa values greater than 10. Thus over a wide pH range from pH > 2, PA is always negatively charged.25 Therefore, phytic acid occupies all six coordination sites of chitosan during the ionic gelation process. In the ionic gelation, the extrusion of liquid through a capillary nozzle is necessary for the formation of capsules. Generally, chitosan-insulin suspension is dropped from a capillary nozzle to the cross-linking agent solution for the formation of capsules. However, capsules are formed with diameters ranging 25 mm, which is too large for many biotechnological or medical applications.26 One of the promising techniques for size control of airborne droplets is the electrohydrodynamic atomization (EHDA) process, also referred to as the electrospray process. In the EHDA process, highly charged, relatively monodisperse droplets that were controlled in size can be produced from various conditions of liquid solution material. Microdripping, which is one of the spray modes, produces a narrow size distribution at a production frequency of a few kHz. An applied electric field transforms the drop hanging from the capillary. The electric field suppresses the surface tension since the electric polarization forces act in the opposite direction of the surface tension. Furthermore, the electric force due to the surface charge and the electric field interaction accelerates the fluid downward in the direction of
gravity and away from the nozzle. The final effect is that a smaller aerosol droplet can be obtained.27,28 The ionic gelation method in combination with EHDA is a better way to produce size-controlled capsules in comparison to the ionic gelation method alone since the capsules with a narrow size distribution are necessary in the drug delivery system in order to precisely determine the dose of the drug.1,29 Therefore, it is worthwhile to develop a methodology for controlling release kinetics employing monodisperse microsized capsules for oral drug delivery. In this paper, microsized airborne chitosaninsulin droplets were produced via the microdripping mode of EHDA. The formation of airborne droplets was monitored by a high-speed camera. The produced airborne droplets were fallen into a beaker and reacted with phytic acid, resulting in chitosan capsules containing insulin. The size distribution of chitosan capsules were measured by an optical microscope for finding relations between capsule mean diameter, capsule standard deviation, and nozzle diameter. Encapsulation efficiency was obtained with various nozzle diameters. Structure and inner component of the chitosan capsules were measured by a scanning electron microscope (SEM) and Fourier transform infrared (FTIR) spectroscopy. In vitro studies of drug release rate from different sized chitosan capsules were performed to determine the drug release profile from the capsules.
Figure 1. Experimental setup.
2. MATERIALS AND METHODS The experimental setup consisted of a liquid supply system, a stirring system, a visual system, and an electrical system, as shown in Figure 1. The liquid supply system consisted of a syringe pump (minimum flow rate = 16.7 pL/min for a 1 mL syringe), a feeding tube, and a stainless steel nozzle. Various sized nozzles were used (outer (inner) diameter =1260 (860), 900 (600), 700 (400), 550 (310), 450 (210), 350 (150), 320 (140) μm). The minimum nozzle size was selected after the test of nozzle clogging. The flow rate was fixed at 0.1 mL/min. The visual system consisting of a high-speed camera (Motion Pro HS-4, Redlake Inc.) and a halogen light source (KLS-100H-RS-150, Kwangwoo Co. Ltd.) was used to measure the diameters of aerosol droplets released
Figure 2. Experimental procedure. 13763
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research
ARTICLE
from the nozzle and thereby monitor the microdripping mode of EHDA. The stirring system was located below the glass vial to prevent aggregation. The electrical system consisted of a highvoltage power supply (∼15 kV direct current (dc)) and two electrodes. The nozzle used for the liquid supply system was used as the anode, and a plate-type electrode located 3 cm below the tip of the nozzle was used as the ground electrode. The diameters of encapsulated particles were measured using an optical microscope (BX60M, Olympus). Chitosan was obtained from Greenbiotech Co., Ltd. (Gyeonggi-do, Korea). Phytic acid (PA) solution was purchased from Mitsui Fine Chemical, Inc. (Tokyo, Japan). Insulin (Apidra) was purchased from Sanofi Aventis (Frankfurt, Germany). The molecular weight of the chitosan sample was measured by a modular summit HPLC (high-performance liquid chromatography) system (Dionex, USA) equipped with a P680 gradient pump, ASI100 autosampler, TCC-100 column oven, and UVD340 detector. Figure 2 shows the experimental procedure using the ionic gelation method in combination with EHDA. Chitosan was added to an acetic acid solution of an acidity of 1% (v/v) so that its final concentration became 3% (w/w), and the mixture was stirred at 300 rpm for 10 min using a magnetic stirrer. Bovine insulin, which is a soluble active ingredient, was dissolved in a 0.1 N HCl solution. The insulin solution was added to the chitosan aqueous solution, stirred at 500 rpm for 10 min using a magnetic stirrer to disperse insulin, and then left alone for a while to remove bubbles from the chitosan solution; thus, a chitosan insulin aqueous solution was obtained. Table 1 shows the ink properties of the chitosaninsulin aqueous solution. The density of the chitosaninsulin aqueous solution was determined using the relationship between the mass and volume of the solution in a bottle. The viscosity and electrical conductivity of the solution were measured using a vibration viscometer (SV-10, AND, Japan) and a conductivity meter (F-54, Horiba, Japan), respectively. Surface tension was measured by the well-known Du Nouy ring method. In every EHDA test, a syringe of 1 mL volume (Hamilton, USA), which contained 2.105 mg of insulin, was used. The prepared chitosaninsulin aqueous solution was injected by a syringe pump, and then sprayed on to the phytic acid aqueous solution, Table 1. Properties of Chitosan and Insulin Suspended Ink density
viscosity
surface tension
electrical conductivity
(kg/m3)
(mPa 3 s)
(dyn/cm)
(mS/m)
996
90.6
69.7
5.31
which was filled in the beaker. The phytic acid solution was prepared by adding the commercial phytic acid solution to distilled water and stirred to regulate the concentration of phytic acid to water (6% v/v), followed by regulating its pH to be 2 using a 5.0 N sodium hydroxide solution. The resulting mixture was reacted to induce binding between chitosan and phytic acid, resulting in the chitosan capsules. After the ionic gelation, the capsules gravitationally settled down, and then the solution was decanted by a pipet. The capsules were separated from the mixture, washed with distilled water, and then freeze-dried. The temperature and duration were maintained at 40 °C for 4 h, at 30 °C for 3 h, 10 °C for 2 h, 0 °C for 1 h, 20 °C for 2 h, and 30 °C for 5 h. The pressure was 510 mTorr. The encapsulation efficiency of the chitosan capsule, η, was calculated by the following equation: η ¼ 1
M M0
ð1Þ
where M0 is the total amount (mg) of insulin in the mixture (=2.105 mg) and M is the amount (mg) of insulin lost in the cross-linking medium of phytic acid aqueous solution. M was measured by HPLC. To examine the bioavailability of the chitosan capsules in the in vitro tests, an artificial gastric juice (pH 1.2) and an artificial intestinal juice (0.2 M KH2PO4 25% (v/v), 0.2 N NaOH 11.8% (v/v) in water, pH 6.8) were prepared. The capsules were put in the artificial gastric juice and shaken at 80 rpm for 2 h in a shaking incubator (37 °C). Then the capsules were put in the artificial intestinal juice and shaken at 80 rpm for 8 h in the shaking incubator (37 °C). The release amount of insulin depending on time was measured for 10 h. The samples at appropriate intervals were withdrawn and assayed by HPLC. The insulin release (%) from the chitosan capsule was calculated by the following equation: Insulin release at time t ð%Þ ¼
mt mt1 100 M0 M
ð2Þ
where t = 0 is the time when the capsule is put in the artificial gastric juice; mt is the amount (mg) of insulin lost at a time t. The surface and cross-sectional morphologies of the dried particles were examined using SEM (JSM-6500F, Jeol Ltd., Japan). Infrared spectra of chitosan capsules with and without insulin were obtained with FTIR spectroscopy (Vertex 70, Bruker Optics, USA). The spectra were scanned over wave numbers within the range of 2000 to 1300 cm1. For each spectrum a
Figure 3. Formation of droplets with various applied voltages (nozzle diameter =1260 μm). 13764
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research
ARTICLE
64-scan interferogram was collected at room temperature in N2 atmosphere.
the dark circle was observed due to the transmission of the light from the lamp, since the drop shadow was projected into a microscope and digitized by the high-speed camera. In the absence of an applied voltage, a liquid droplet continued to grow until gravitational force overcame the surface tension. When gravity was the only force acting on the meniscus of the droplet, large sized droplets were produced. Under the action of an electric field, the electric force acting along with the gravitational force reduced the critical volume for drop detachment resulting in a smaller droplet diameter. As the applied voltage increased from 0 to 5 kV, the droplet size decreased. When the voltage changed from 5 to 6 kV, however, the secondary droplet was generated and the size of the primary droplet dramatically decreased. For any voltage between 6 and 9 kV, each droplet was divided into a primary droplet and a secondary droplet, and the primary droplet size decreased as the voltage increased. For voltages higher than 9 kV, the primary droplet size did not change. The results shown in Figure 3 are summarized in Figure 4, which also show the calculated results. The theoretical droplet diameter, dd, was obtained assuming a condition for mechanical equilibrium of a droplet in an electric field under the voltage difference between the nozzle and ground, V27
3. RESULTS AND DISCUSSION Figure 3 shows magnified images of airborne droplets released from a capillary nozzle for various applied voltages from 0 to 11 kV. The location of the airborne droplets was about 12 cm below the end of the capillary nozzle. The nozzle diameter was 1260 μm. The dark circle represents the cross-section chitosaninsulin aqueous solution droplet. The small white spot at the center of
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u " 2 # u 3 6 1 4V 2dN γ dN 2 ε0 dd ¼ t Fg 4 dN lnð8H=dN Þ
Figure 4. Diameters of primary droplets with various applied voltages (nozzle diameter =1260 μm).
ð3Þ
Figure 5. Droplet formation in the microdripping mode (applied voltage = 9 kV).
Figure 6. Droplets with various nozzle diameters (applied voltage = 9 kV). 13765
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research where ε0 is the permittivity of air (8.859 1012 F/m), H is the distance between the nozzle and the ground, γ is the surface tension, and dN is the diameter of nozzle. For lower applied voltages, the theoretically predicted values matched the experimental data. However, for higher applied voltages, they provided a poorer fit to the experimental data. The deviations between the experimental and calculated values might be due to the missing contributions of the solution flow rate and solute concentration,27 in addition to secondary atomization. Generally, the transition from the microdripping mode to the cone-jet mode can further reduce the droplet size with the increase of the applied voltage. Enayati et al.30,31 and Chang et al.3234 reported that the cone-jet mode can produce much smaller droplets than the microdripping mode. In this work, however, such a transition did not occur due to the high surface tension and viscosity of the liquid solution. Generally, the ranges of the viscosity and conductivity of the solution for the cone-jet mode have been reported as 0.2993 mPa 3 s and 103108 S/m, respectively (see Table 1).3537 Stachewicz et al.38 mentioned that the EHDA of water, especially in the cone-jet mode, was difficult due to the high surface tension of water. To overcome the high surface tension and to achieve a stable cone-jet mode of water, a high electric field would be required, which, however, might cause a spark discharge. In this study, when the voltage of 12 kV was applied, the spark discharge was occurred since the chitosan and insulin suspended ink was water-based. Therefore, voltages below 12 kV were applied to avoid the spark discharge.
Figure 7. Primary droplet diameters with various nozzle diameters (applied voltage = 9 kV).
ARTICLE
In the following experiments, the applied voltage was fixed at 9 kV since the droplet diameter did not change much for voltages above 9 kV. Figure 5 shows photos of the droplets taken at various time steps. Shortly after the lower part of a hanging drop began to fall, it produced the tail on which surface tension acted, making it thinner. At a certain stage, the long tail was pinched off from both sides and eventually contracted into a secondary droplet. The whole sequence of the pictures was completely reproducible. According to Eggers,39 a liquid with high viscosity and high surface tension could result in a long and thin tail in the microdripping mode. The formation of droplets was investigated with a visual system for various nozzle diameters. The applied voltages were 9 kV for all cases. Figure 6 shows that as the nozzle diameter decreased, the sizes of the primary drop as well as the secondary droplet also decreased. The results are summarized in Figure 7. The standard deviation of the primary droplet size decreased as the nozzle diameter decreased. Once the droplet aerosols arrived at a liquid bath containing the phytic acid aqueous solution, the droplet aerosols were transformed into capsules containing insulin. After the freeze-drying method, every capsule was fixed in a jig, and then the crosssection of the capsule was cut by a sharp razor. A conductive carbon tape was used for mounting the cross-sectioned or the original capsules. Then, a platinum coating process was achieved. Figure 8a shows an SEM image for the cross-section of a chitosan capsule. The hydrogel matrix of the chitosan and the dispersed insulin was wrapped by the shell. Figure 8b is a magnified image
Figure 9. FTIR spectra of chitosan capsule without and with insulin.
Figure 8. SEM images of chitosan capsules (a) cross-section of capsule and (b) its inner surface. 13766
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research
ARTICLE
Figure 10. Size distribution of capsule diameter with various nozzle diameters (applied voltage = 9 kV) (a) 1260, (b) 900, (c) 700, (d) 550, (e) 450, (f) 350, (g) 320 μm.
of the hydrogel matrix from which the distinction between the chitosan and the insulin was not apparent. Therefore, FTIR spectra of the hydrogel matrix were obtained (see Figure 9) to detect any shift in peaks that could be attributed to interactions between the insulin and the chitosan. Pellets for FTIR spectra were prepared by grinding the insulin powder and the chitosan capsules with and without insulin. The grinded powders were mixed with solid potassium bromide (KBr) since KBr is transparent to infrared radiation. For comparison, FTIR spectra of encapsulated chitosan not containing insulin are plotted in Figure 9. In addition, FTIR spectra of bovine insulin powder are presented. Saramento et al.40 also observed the shift of peaks
in FTIR spectra of alginate/chitosan powder and insulin-loaded alginate/chitosan powder. The results of Figure 9 are shown in terms of transmittance, T, which is defined as TðλÞ ¼
IðλÞ I0 ðλÞ
ð4Þ
where I0(λ) is the intensity of the light source beam of wavelength λ and I(λ) is the intensity of the beam after passing through the capsule at the wavelength of λ. The FTIR spectra of a capsule without insulin show two peaks (∼1636 and 1541 cm1). 13767
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research However, the FTIR spectra of the capsule containing insulin were shifted to 1638 and 1535, which are closer to those of insulin powder, 1657 and 1535 cm1. According to previous works,4143 two shoulders on the transmittance bands at wave numbers of ∼1657 and ∼1535 cm1 are characteristics of protein spectra in the amide I and amide II groups, respectively. Insulin is one of the proteins. Brange44 stated that the insulin monomer contains many ionizable groups, due to 6 amino acid residues capable of attaining a positive charge and 10 amino acid residues capable of attaching a negative charge. The capsules with and without insulin were formed by the ionic gelation between positively charged chitosan and negatively charged phytic acid. For every nozzle diameter, a sample of 100 individual chitosan capsules was randomly chosen and their sizes were measured by an optical microscope. The applied voltage was fixed at 9 kV. Figure 10 panels ag show size distributions of chitosan capsules with different nozzle diameters. Photos of the bath surface where the capsules are shown as dots are listed as insets of Figure 10. The capsule diameter was defined as a volume equivalent diameter since the capsules had nonspherical shapes. The mean diameters of the capsules were 421, 415, 412, 410, 257, 239, 232 μm (standard deviation = 292, 255, 246, 188, 101, 69, 67 μm) for 1260, 900, 700, 550, 450, 350, 320 μm nozzle diameters, respectively. The mean diameters of the capsules were much smaller than those of the primary droplets since moisture in the capsules were removed during the drying process. The large standard deviations were caused by the capsules formed from the secondary droplets in addition to the primary droplets. According to these experimental data, the following relationship was obtained. pffiffiffiffiffiffiffiffiffiffiffi ð5Þ dmean, capsule ≈ dnozzle These experimental results showed that smaller and more monodispersed capsules were generated for smaller nozzles. Chitosan was polycationic in the acidic aqueous solution and interacted with negatively charged species such as phytic acid. The interaction of chitosan with phytic acid led to the formation of the capsules, which could be efficiently employed in insulin delivery. Figure 11 shows the encapsulation efficiency, as described in eq 1, with various nozzle diameters. The encapsulation efficiencies of PA/chitosan microcapsules were 9297%, which were slightly dependent on the nozzle diameter. Capsule size significantly impacts critical product characteristics such as dissolution rate and dosage unit content uniformity.45 Oral insulin released from encapsulated capsules needs to be controlled and sustained. Preferably, the encapsulated capsules must reach the small intestine in the gastro-intestinal tract. The pH of the gastro-intestinal juice in the local region of the intestine influences a drug’s dissolution rate and possibly its permeability. The pH strongly influences the solubility of the capsules by determining their ionization state. When the pH is such that the chitosan capsules are in its ionic form, the solubility of the capsule is usually high compared to its nonionized form. The pH thus has a strong effect on the dissolution of the chitosan capsules. An in vitro test was conducted when the capsules were put in an artificial gastric juice (pH 1.2). After 2 h, the capsules were moved to an artificial intestinal juice and further in vitro tests were performed for 8 h. Figure 12 shows the variation of insulin release (%) with respect to time for different nozzle diameters. The amounts of insulin release were sampled after 2, 3, 4, 6, 8, 10 h and measured
ARTICLE
Figure 11. Encapsulation efficiency with various nozzle diameters.
Figure 12. Insulin release with various nozzle diameters.
by the HLPC. Then the insulin release (%) was calculated using eq 2. Figure 12 indicates that the size of the capsule is an important factor that affects drug dissolution. For any nozzle size, Figure 12 demonstrated a burst release during 2 h because of an acidic condition in the artificial gastric juice. The release rate of smaller capsules (corresponding to smaller nozzles) was relatively higher than that of larger capsules (corresponding to larger nozzles) in the artificial gastric juice since smaller capsules might have a lower resistance to acidic conditions, which was also stated by Pan et al.46 Decreasing capsule size, thus increasing surface area, speeds up the dissolution of the capsule, which was also observed by Dressman et al.47 during modeling. The insulin release from 2 to 4 h decreased dramatically when the capsules were forced to move from the artificial gastric juice to the artificial intestinal juice. However, the insulin that was released between 4 to 6 h had increased, except for the case in which the nozzle diameter was 1260 μm in nozzle. This insulin release was constant for all nozzle sizes. The dissolution test showed that when a nozzle smaller than or equal to about 700 μm was used, the sustained insulin release occurred in the artificial intestinal juice although the smaller capsules exhibited lower resistance to acidic conditions.
4. CONCLUDING REMARKS Microsized airborne droplets containing insulin were produced via the microdripping mode of EHDA by chitosan insulin (mass ratio of 9:1) aqueous solution. The produced airborne chitosaninsulin droplets were reacted with phytic acid 13768
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research to induce the binding between chitosan and phytic acid, resulting in chitosan capsules. As the nozzle size decreased, the size and uniformity of the primary airborne droplets decreased and enhanced, respectively. The size of the airborne droplets also affected the uniformity of the capsules’ size. The mean diameter of the capsules was proportional to the square root of the nozzle diameter. The mean diameter was 232 μm for a nozzle with a diameter of 320 μm, which was much smaller than that of the capsules formed using the ionic gelation method alone. The insulin encapsulation efficiency was nearly independent of the nozzle size and was above 92%. Through in vitro tests carried out with the capsules, when a nozzle smaller than or equal to about 700 μm was used, the sustained insulin release occurred in the artificial intestinal juice although the smaller capsules exhibited lower resistance to acidic conditions. However, in order to clarify the effect of capsule size on their absorption in the gastrointestinal tract of animals, further study is needed using in vivo tests with various sized chitosan capsules containing insulin in order to understand efficacies for the cure of diabetic symptoms.
’ AUTHOR INFORMATION Corresponding Author
*Tel: +82-2-2123-2821. Fax: +82-2-312-2821. E-mail: hwangjh@ yonsei.ac.kr.
’ ACKNOWLEDGMENT This work was supported by the Global Frontier R&D Program on Center for Multiscale Energy System funded by the National Research Foundation under the Ministry of Education, Science and Technology, Korea. ’ REFERENCES (1) Gholamipour-Shirazi, A. Insulin encapsulation. Expert Opin. Drug Delivery 2008, 5, 1335. (2) Carino, G. P.; Mathiowitz, E. Oral insulin delivery. Adv. Drug Delivery Rev. 1999, 35, 249. (3) des Rieux, A.; Fievez, V.; Garinot, M. Nanoparticles as potential oral delivery systems for proteins and vaccines: A mechanistic approach. J. Controlled Release 2006, 116, 1. (4) Silva, G. A.; Ducheyne, P.; Reis, R. L. Material in particulate form for tissue engineering. J. Tissue Eng. Regen. Med. 2007, 1, 4. (5) Pancholi, K.; Ahras, N.; Stride, E.; Edirisinghe, M. Novel electrohydrodynamic preparation of porous chitosan particles for durg delivery. J. Mater. Sci. 2009, 20, 917. (6) Denkbas, E. B.; Seyyal, M.; Piskin, E. 5-Fluorouracil loaded chitosan microspheres for chemoembolization. J. Microencap. 1998, 16, 741. (7) Sankar, C.; Rani, M.; Srivastava, A. K.; Mishra, B. Chitosan based pentazocine microspheres for intranasal systemic delivery: Development and biopharmaceutical evaluation. Pharmazie 2001, 56, 223. (8) Jameela, S. R.; Kumary, T. V.; Lal, A. V.; Jayakrishnan, A. Progesterone-loaded chitosan microspheres: A long acting biodegradable controlled delivery system. J. Controlled Release 1998, 52, 17. (9) Berthod, A.; Kreuter, J. Chitosan microspheres-improved acid stability and change in physicochemical properties by cross-linking. Proc. Int. Symp. Control Rel. Bioact. Mater. 1996, 23, 369. (10) Turan, S. O.; Akbuga, J.; Aral, C. Controlled release of interleukin-2 from chitosan microspheres. J. Pharm. Sci. 2002, 91, 124. (11) Mao, H. Q.; Roy, K.; Le, V. L.; Janes, K. A.; Kim, K. Y.; Wang, Y.; August, J. T.; Leong, K. W. Chitosan DNA nanoparticles as gene delivery carriers: Synthesis, characterization and transfection efficiency. J. Controlled Release 2001, 70, 399.
ARTICLE
(12) He, P.; Davis, S. S.; Illum, L. Chitosan microspheres prepared by spray drying. Int. J. Pharm. 1999, 187, 53. (13) Conti, B.; Modena, T.; Genta, I.; Perugini, P.; Decarro, C.; Pavanetto, F. Microencapsulation of cetylpyridinium chloride with a bioadhesive polymer. Proc. Int. Symp. Control Rel. Bioact. Mater. 1998, 25, 822. (14) Huang, C.; Yeh, M. K.; Chiang, C. H. Formulation factors in preparing BTM-chitosan microspheres by spray drying method. Int. J. Pharm. 2002, 242, 239. (15) Shi, X. Y.; Tan, T. W. Preparation of chitosan/ethlcellulose complex microcapsule and its application in controlled release of vitamin D-2. Biomaterials 2002, 23, 4469. (16) Shu, X. Z.; Zhu, K. J. Chitosan/gelatin microspheres prepared by modified emulsification and ionotropic gelation. J. Microencap. 2001, 18, 237. (17) Mi, F. L.; Sung, H. W.; Shu, S. S. Release of indomethacin from a novel chitosan microsphere prepared by naturally occurring crosslinker: Examination of crosslinking and polycation/anion drug interaction. J. Appl. Polym. Sci. 2001, 81, 1700. (18) Ko, J. A.; Park, H. J.; Hwang, S. J.; Park, J. B.; Lee, J. S. Preparation and characterization of chitosan microparticles intended for controlled drug delivery. Int. J. Pharm. 2002, 249, 165. (19) Calvo, P.; Lopez, C. R.; Jata, J. L.; Alonso, M. J. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J. Appl. Polym. Sci. 1997, 63, 125. (20) Shu, X. Z.; Zhu, K. J. A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery. Int. J. Pharm. 2000, 201, 51. (21) Urrusuno, R. F.; Cavlo, P.; Lopez, C. R.; Jato, J. L.; Alonso, M. J. Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm. Res. 1999, 16, 1576. (22) Pan, Y.; Li, Y.; Zhao, H.; Zheng, J.; Xu, H.; Wei, G.; Cui, F. Chitosan nanoparticles improve the intestinal absorption of insulin in vivo. Int. J. Pharm. 2002, 249, 139. (23) Wu, L. Q.; Gadre, A. P.; Yi, H.; Kastantin, M. J.; Rubloff, G. W.; Bentley, W. E.; Payne, G. F.; Ghodssi, R. Voltage-dependent assembly of the polysaccharide chitosan onto an electrode surface. Langmuir 2002, 18, 8620. (24) Tsai, M. L.; Bai, S. W.; Chen, R. H. Cavitation effects versus stretch effects resulted in different size and polydispersity of ionotropic gelation chitosansodium tripolyphosphate nanoparticle. Carbohydr. Polym. 2008, 71, 448. (25) Yang, L.; Liu, H.; Hu, N. Assembly of electroactive layer-bylayer films of myoglobin and small-molecular phytic acid. Electrochem. Commun. 2007, 9, 1057. (26) Heinzen, C.; Berger, A.; Marison, I. Use of vibration technology for jet break-up for encapsulation of cells, microbes and liquids in monodisperse microcapsules. Landbauforsch Volkenrode 2002, 241, 19. (27) Xie, J.; Wang, C. H. Electrospray in the dripping mode for cell microencapsulation. J. Colloid Interface Sci. 2007, 312, 247. (28) Hartman, R. P. A. Electrohydrodynamic atomization in the cone-jet mode. Ph.D. Thesis, Delft University, The Netherlands, 1998. (29) Arya, N.; Charkraborty, S.; Dube, N.; Katti, D. S. Electrospraying: A facile technique for synthesis of chitosan-based micro/nanospheres for drug delivery applications. J. Biomed. Mater. Res., Part B 2009, 88, 17. (30) Enayati, M.; Ahmad, Z.; Stride, E.; Edirisinghe, M. One-step electrohydrodynamic production of drug-loaded micro- and nanoparticles. J. R. Soc. Interface 2010, 7, 667. (31) Enayati, M.; Ahmad, Z.; Stride, E.; Edirisinghe, M. Preparation of polymeric carriers for drug delivery with different shape and size using an electric jet. Current Pharm Biotechnol. 2009, 10, 600. (32) Chang, M. W.; Stride, E.; Edirisinghe, M. A new method for the preparation of monoporous hollow microspheres. Langmuir 2010, 26, 5115. (33) Chang, M. W.; Stride, E.; Edirisinghe, M. Controlling the thickness of hollow polymeric microspheres prepared by electrohydrodynamic atomization. J. R. Soc. Interface 2010, 7, S451. 13769
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
Industrial & Engineering Chemistry Research
ARTICLE
(34) Chang, M. W.; Stride, E.; Edirisinghe A novel process for drug encapsulation using a liquid to vapour phase change material. Soft Matter 2009, 5, 5029. (35) Zhang, H. B.; Jayasinghe, S. N.; Edirisinghe, M. J. Electrically forced microthreading of highly viscous dielectric liquids. J. Electrostat. 2006, 64, 355. (36) Barrero, A.; Lopez-Herrera, J. M.; Boucard, A; Loscertales, I. G.; Marquez, M. Steady cone-jet electrosprays in liquid insulator baths. J. Colloid Interface Sci. 2004, 272, 104. (37) Jayasinghe, S. N.; Edirisinghe, M. J.; Wang, D. Z. Controlled deposition of nanoparticle clusters by electrohydrodynamic atomization. Nanotechnology 2004, 15, 1519. (38) Stachewicz, U.; Yuteri, C. U.; Dijksman, J. F.; Marijnissen, J. C. M. Single event electrospraying of water. J. Aerosol Sci. 2010, 41, 963. (39) Eggers, J. Nonlinear dynamics and breakup of free-surface flows. Rev. Mod. Phys. 1997, 69, 865. (40) Sarmento, B.; Ferreira, D.; Veiga, F.; Ribeiro, A. Characterization of insulin-loaded alginate nanoparticles produced by ionotropic pregelation through DSC and FTIR studies. Carbohydr. Polym. 2006, 66, 1. (41) Wei, J.; Lin, Y. Z.; Zhou, J. M.; Tsou, C. L. FTIR studies of secondary structures of bovine insulin and its derivatives. Biochim. Biophys. Acta 1991, 1080, 29. (42) Xie, L.; Tsou, C. L. Comparison of secondary structures of insulin and proinsulin by FTIR. J. Protein Chem. 1993, 12, 483. (43) Dzwolak, W.; Ravindra, R.; Lendermann, J.; Winter, R. Aggregation of bovine insulin probed by DSC/PPC calorimetry and FTIR spectroscopy. Biochemistry 2003, 42, 11347. (44) Brange, J. Galenics of insulin: The Physic-Chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations; Springer: Berlin, 1987. (45) Rohrs, B. R.; Amidon, G. E.; Meury, R. H.; Secreast, P. J.; King, H. M.; Skoug, C. J. Particle size limits to meet USP content uniformity criteria for tablets and capsules. J. Pharm. Sci. 2006, 95, 1049. (46) Pan, Y.; Zheng, J. M.; Zhao, H. Y.; Li, Y. J.; Xu, H.; Wei, G. Relationship between drug effects and particle size of insulin-loaded bioadhesive microspheres. Acta Pharmacol. Sin. 2002, 23, 1051. (47) Dressman, J. B.; Amidon, G.; Reppas, C.; Shah, V. P. Dissolution testing as a prognostic tool for oral drug absorption: immediate release dosage forms. Pharm. Res. 1998, 15, 11.
13770
dx.doi.org/10.1021/ie200915x |Ind. Eng. Chem. Res. 2011, 50, 13762–13770
