Pharmaceutical Chemistry Journal
Vol. 44, No. 10, 2011
PECTIN–ZEIN MICROSPHERES AS DRUG DELIVERY SYSTEMS Z. K. Mukhidinov,1 G. F. Kasimova,1 D. T. Bobokalonov,1 D. Kh. Khalikov,1 Kh. I. Teshaev,1 M. D. Khalikova,1 and L.-S. Liu2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 44, No. 10, pp. 35 – 39, October, 2010. Original article submitted July 9, 2009.
The formation of microspheres from various pectin hydrogel complexes and corn zein in the presence of calcium and zinc ions has been studied. It is shown that the formation of microspheres and their loading capacity for a drug (piroxicam) depend on the type of biopolymers, their ratio, the sizes of the bivalent ions, and the molecular mass of the pectin. Complex formation between the two biopolymers results predominantly from bivalent metal cross-linking for low-methylated pectins and from hydrophobic interaction for high-methylated pectins. As a result, a series of microspheres have been prepared from biodegradable and biocompatible polymers and may find application as controlled-release drug delivery systems. Key words: pectin, zein, microspheres, drug delivery systems.
Delivery systems that target drugs to a certain body region have recently been widely used in medicine, the pharmaceutical industry, and cosmetology. Polysaccharides and proteins are interesting for creating biocompatible drug delivery systems that are stable to the action of enzymes in the GI tract. Pectin is a promising biopolymer that is designed for treating various intestinal diseases. The ability of low-methylated (LM) pectin to form a gel in the presence of metal ions provided a basis for creating drug delivery systems in the form of microspheres, microcapsules, and compositions [1, 2]. However, the high degree of swelling of pectin gel under physiological conditions led to premature degradation as a result of the expansion of its pores. This process could be prevented by increasing the water-resistance of the pectin, which can be achieved in combination with hydrophobic materials. An alcohol-soluble protein from corn, zein, is widely used in the food and pharmaceutical industries because of its hydrophobic nature and ability to form films. It was supposed that complexes of pectin with zein would suppress pectin swelling in the stomach and, thereby, limit drug degradation in the GI tract [3]. The aim of the present study was to develop conditions for preparing pectin/zein (Z/P) complexes in the form of 1 2
microspheres with encapsulated drugs as controlled-release drug delivery systems in the intestines. EXPERIMENTAL PART We used pectins isolated from the following sources: LM-31 citrus pectin (GENU 12CG-CP Kelco), degree of esterification (DE) 31%, galacturonic acid (GA) content 69.0%, MW 308 kDa; LM-9 (GENU L/200-CP Kelco), DE 9%, GA 69.6%, MW 466 kDa; HM apple pectin (Muminobad Region, Tajikistan), DE 52%, GA 68%, MW 134 kDa; and LM apple pectin (Shakhrinau Region, Tajikistan), DE 40,3%, GA 58.3%, MW 18.7 kDa. Zein was isolated from defatted corn flour by the published method [4]. The nonsteroidal anti-inflammatory drug piroxicam was used as the model drug. Z/P microspheres were formed using two methods: a) by adding pectin solution (12 mL) to an alcoholic solution of zein (75 vol %, 15 mL) containing piroxicam and CaCl2 (calculated for 30 mg Ca2+ per g of pectin) at room temperature with stirring and b) by adding dropwise an aqueous pectin solution (12 mL) containing piroxicam in alcohol (3 mL, 75 vol %) to an alcoholic solution of zein (13 mL, 75 vol %) and bivalent metal salts (CaCl2, ZnSO4) with piroxicam and the cross-linking metals taken twice greater. The zein content was varied from 50 to 1000 mg; pectin, from 200 to 350 mg; piroxicam, from 20 to 50 mg in all experiments with the ex-
Institute of Chemistry, Tajik Academy of Sciences, Dushanbe, Tajikistan Republic. Eastern Regional Research Center, U. S. Department of Agriculture, Philadelphia, USA.
564 0091-150X/11/4410-0564 © 2011 Springer Science+Business Media, Inc.
Pectin–Zein Microspheres as Drug Delivery Systems 100
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2 80 1
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Mass ratio
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1
60 40 20
20
3
3 0
0 0
1
2
3 Z/P ratio
4
5
6
0
0.5
1.0 Z/P ratio
1.5
2.0
Fig. 1. Microsphere yield, degree of swelling, and piroxicam content as functions of zein/pectin ratio: complex yield (1 ), piroxicam content in complex (2 ), degree of complex swelling (3 ).
Fig. 2. Microcapsule yield in the presence of Ca2+ ions, degree of swelling, and piroxicam content as functions of zein/pectin ratio: complex yield (1 ), piroxicam content in complex (2 ), degree of complex swelling (3 ).
ception of the complex with Z/P = 4:1, where piroxicam was taken in an excess (200 mg). The resulting complexes were washed with water and EtOH (50 vol %) in order to remove non-bound components and were dried at 25 – 30°C to constant mass. Piroxicam bound in complexes was determined by treating dry compound (15 mg) with EtOH (10 mL, 75 vol %) containing NaOH (0.4 mass %) and Tween-20 (0.5 mass %) and thermostatting at 37°C overnight. The total piroxicam content in the supernatant after centrifuging was determined using a calibration curve constructed using pure piroxicam and a Thermo Spectronic UV-1 spectrophotometer (UK) at 355 nm. Table 1 lists the initial ratios of principal components used to form microspheres with LM-31 citrus pectin and their properties such as complex yield, degree of swelling, and amount of bound piroxicam. Figure 1 shows the yield of microspheres, their swelling, and the content of encapsulated piroxicam as functions of the Z/P ratio. The results indicate that the yield of microcapsules produced from LM-31 pectin and the degree of swelling of piroxicam complexes depend on the Z/P ratio. The greater
the amount of zein in the complex is, the greater the degree of drug binding. At all Z/P ratios, the effectiveness of the encapsulation increases from 9.8 to 93.8 mass % with increasing zein content from 1 to 5. It should also be noted that the desired effect was not achieved upon increasing the amount of piroxicam by 10 times with Z/P = 1.4:1. Apparently this was due to the nature of the biopolymers and the packing density of the hydrophobic parts of the polymeric chain. In this instance it could be confirmed that enthalpy factored into the formation of the complexes. It is noteworthy that the nature of the biopolymers; their ratio, molecular mass, and charge density; and several other factors affected the properties of the Z/P complexes. The Z/P complexes formed through ionic and hydrogen bonds between the protein and polysaccharide whereas hydrophobic interactions were insignificant. Turbidimetric titration [5] in the presence of Ca2+ ions showed that all zein was incorporated into the complex as the CaCl2 concentration increased. However, two types of complexes formed as the zein concentration increased. These were pectin—Ca2+—zein and pectin—zein. The first type might have formed as a result of electrostatic interactions due to cross-linking of Ca2+ with pectin chains; the second,
TABLE 1. Initial Component Ratios for Preparing LM-31 Zein/Pectin Microspheres and Their Properties Z/P
Zein, mg
Pectin, mg
CaCl2, mg
Piroxicam, mg
Yield, mass %
Degree of swelling
Piroxicam in complex, %
1:1 1:3 1:6 1.4:1 1.4:1 2:1 5:1
250 100 50 500 500 500 1000
250 300 300 350 350 250 200
20.6 24.8 24.8 20.6 20.6 20.6 16.6
20 20 20 20 200 20 20
37.0 15.6 16.5 45.0 55.6 55.6 64.7
49.0 56.1 68.0 33.25 13.91 19.45 7.5
37.0 9.8 12.4 57.0 51.7 72.6 93.8
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100
80
2
80
2
Mass ratio
Mass percent
70 60
60
40
1
50 20
40
3
1 30
0 0
1
2 Ca2+, %
3
4
Fig. 3. Effect of amount of Ca2+ ions on yield of hydrogel microspheres and content of encapsulated piroxicam; LM-9 citrus pectin; Z/P ratio = 1:1: complex yield (1 ) and piroxicam in complex (2 ).
through weak hydrophobic interactions and coacervation of the two biopolymers. An important indicator for effective encapsulation of the drug is the degree of swelling of the complexes. The experimental results showed (Fig. 1) that the degree of swelling depended on the Z/P ratio in the complex. The greater the Z/P ratio was, the less the degree of swelling was. For this, the mass of the complexes increased noticeably. Complexes with Z/P = 1:6 and 1:3 were observed to have a high degree of swelling. It is assumed that these complexes will exhibit kinetics of premature degradation with release of the drug into the GI tract. The optimum Z/P ratio for effective encapsulation was considered to be 5:1. It should be noted that the encapsulated drug should be unaffected in the upper part of the GI tract and the hydrogel
TABLE 2. Z/P Hydrogel Microcapsules Based on LM-9 Citrus Pectin and Their Properties Z/P, mass ratio
2:1 1:3 1.5:1 1:1 1:1 1:1 1:3 1:2 1.5:1 2:1
M2+, % Ca2+
Zn2+
Piroxicam, mg
1.85 1.08 1.27 1.08
50 50 50 50 50 20 50 50 50 50
3.70 3.56 2.02 1.26 2.44 2.57
Micro- Degree capsule of yield, % swelling
66.70 64.20 46.60 34.50 76.30 35.70 61.20 28.60 45.00 34.50
13.69 29.10 21.00 11.50 10.33 11.00 31.10 19.50 16.00 5.80
Piroxicam content in complex, %
75.80 81.80 95.20 66.00 66.00 53.30 86.00 52.80 80.60 80.60
0
0.5
1.0
1.5 Z/P ratio
2.0
2.5
Fig. 4. Microsphere yield, degree of swelling, and piroxicam content as functions of Z/P ratio; LM-9 citrus pectin, cross-linking Zn2+: complex yield (1 ), piroxicam in complex (2 ), degree of complex swelling (3 ).
matrix should be protected from the action of proteases in order to deliver the drug to the large intestine. We modified the experiments by changing the Z/P ratio, the concentration of cross-linking metals and piroxicam, and the technique for preparing the complexes in order to obtain microcapsules with drug that were stable in the upper part of the GI tract. For this, complexes were formed by adding a pectin solution containing piroxicam to zein in alcohol (75 vol %) containing CaCl2 (ZnSO4) with piroxicam and the cross-linking metals taken twice greater. We used pectin isolated from three sources, LM-9 citrus pectin and HM and LM apple pectins. The piroxicam content in the microcapsules was determined by sequential extraction by EtOH (75 vol %) containing NaOH (0.4 mass %), Tween-20 (0.5 mass %), and phosphate buffer (pH 6.4). Tables 2 and 3 list the content of principal components (pectin, zein, CaCl2, ZnSO4) and the properties of the resulting complexes with encapsulated piroxicam. The structure of the produced microspheres differed depending on the properties of the pectins. Well-formed spherical hydrogel microspheres were obtained with LM-9 citrus pectin and HM apple pectin. Figure 2 illustrates the change of the principal parameters of the hydrogel microspheres obtained from LM-9 citrus pectin. It can be seen that the yield of complexes and the content of piroxicam reach maxima for a high content of one of the biopolymers. These parameters pass through a minimum for equal amounts of them. In this instance the degree of swelling of the microspheres decreases with increasing mass fraction of zein, like for complexes with LM-31 citrus pectin. Figure 3 shows the effect of the amount of Ca2+ ions in the microspheres with Z/P = 1:1 on the yield and content of adsorbed piroxicam. It can be seen that the complex yield is
Pectin–Zein Microspheres as Drug Delivery Systems
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TABLE 3. Z/P Hydrogel Microcapsules Based on LM and HM Apple Pectin and Their Properties Z/P, mass ratio
Pectin type
1:2 1:1
HM apple pectin
6:1
-²LM apple pectin
1:2 1:1
-²-
-²-
1:1
-²-
6:1
-²-
M2+, %
Piroxicam, mg
Microsphere yield, %
Degree of swelling
Piroxicam content in microspheres, %
1.78 2.57
20 20
26.50 36.30
26.14 27.,74
58.75 32.50
3.52
20
43.90
30.16
39.00
1.78 2.57
20 20
30.90 33.90
5.25 8.94
66.00 61.10
2+
Ca
Zn2+
1.62 3.52
maximum for a Ca2+ content of 2.02% whereas piroxicam is adsorbed more if the amount of Ca2+ ions is increased to 2.44%. The results indicate that the optimum yield of complexes and a large amount of encapsulated drug can be obtained by regulating the content of Ca2+ ions. Replacing Ca2+ ions by Zn2+ ions (Z/P = 1.5 and 3) causes an insignificant decrease in the mass of the hydrogel microspheres whereas the degree of piroxicam saturation at Z/P = 1.5 decreases from 95 to 80 mass %. However, increasing the pectin fraction (Z/P = 1:3) causes an increase from 81 to 86 mass % (Fig. 4). In this instance, it can be seen that the curves changed in the same way as in Fig. 2 with the exception of the degree of swelling. The results confirm the hypothesis that Zn2+ ions are more effective than Ca2+ ions as cross-linkers that enhance the formation of a more compact structure with pectin. This is important for creating drug delivery systems that are stable toward degradation in the upper part of the GI tract [6]. Z/P microspheres with LM and HM apple pectin with Z/P = 1:6, 1:1, and 2:1 were prepared under analogous conditions. Table 3 shows that the complexes with HM citrus pectin had a greater mass and degree of swelling. However, the degree of piroxicam saturation was greater for the complexes with the LM pectin. It is obvious from the results that increasing the content of Ca2+ ions in the reaction mixture increased the yield of microspheres. Replacing Ca2+ ions by Zn2+ ions increased noticeably the degree of swelling of drug microspheres for Z/P = 1:1. The yield decreased slightly. This confirms that complexes of a different structure were formed. It can be seen that the amount of captured drug passes through a minimum and then increases upon increasing the zein fraction in the complex, the same as in the preceding instances, although the yield of LM apple pectin microspheres decreases noticeably. However, the yield of microspheres and the piroxicam content for apple pectin microspheres were almost half that of LM-9 citrus pectin microspheres. Structurally fragile hydrogel spheres that then transformed into aggregates formed from LM apple pectin in the
20
25.00
16.71
71.50
20
29.30
22.70
70.20
presence of bivalent metal ions. However, the degree of piroxicam binding in the complexes was greater than that for HM pectin. As already noted, these differences were related to structural features of the polysaccharide that formed with zein coacervates capable of capturing a large amount of drug. Thus, the optimum conditions for preparing microspheres from the natural biopolymers pectin and zein with encapsulated drug were determined. It was shown that the nature of the biopolymers, their ratio, the presence of bivalent metals, and the molecular mass of the pectin affect the formation process of the complexes and the degree of saturation of the microspheres by the drug. Microspheres formed using the LM pectins contained complexes formed mainly through cross-linking bonds with bivalent metal ions; using HM pectin, through hydrophobic interactions of the biopolymers. As a result, series of microspheres based on the biodegradable and biocompatible polymers pectin and zein were prepared and may find application in formulating controlled-release drug delivery systems. Kinetic studies of drug release in the GI tract are needed in order to determine the stability of the prepared complexes under physiological conditions. The results of the kinetic studies will be reported separately. REFERENCES 1. L. S. Liu, M. L. Fishman, and K. B. Hicks, Biomaterials, 24, 3333 – 3343 (2003). 2. L. S. Liu, M. Kende, G. Ruthel, et al., Drug Delivery, 13, 417 – 423 (2005). 3. Z. Muhiddinov, D. Khalikov, T. Speaker, and R. Fassihi, Microencapsul, 21(7), 729 – 741 (2004). 4. G. F. Kasimova, D. T. Bobokalonov, M. D. Khalikova, et al., Izv. Akad. Nauk Resp. Tadzh., Otd. Fiz.-Mat., Khim. Geol. Nauk, 2(127), 42 – 50 (2007). 5. G. F. Kasimova, Z. K. Muhidinov, A. Sh. Shtanchaev, et al., Int. Conf. Nanostructures in Polysachcarides, Tashkent, Uzbekistan (2008), pp. 61 – 63. 6. V. Pillay, M. P. Danckwerts, Z. K. Muhydinov, and R. Fassihi, Drug. Dev. Ind. Pharm., 31, 191 – 207 (2005).