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Stearic Acid-Coated Chitosan-Based Interpenetrating Polymer Network Microspheres: Controlled Release Characteristics Sudha C. Angadi, Lata S. Manjeshwar,* and Tejraj M. Aminabhavi† Department of Chemistry, Karnatak University, Dharwad 580 003, India ABSTRACT: Novel pH-sensitive stearic acid-coated interpenetrating polymer network (IPN) blend microspheres of chitosan and gelatin were prepared by the emulsion cross-linking method using glutaraldehyde for the controlled release (CR) of isoniazid (INH), an antituberculosis drug. Coated as well as uncoated microspheres were developed and characterized by Fourier transform infrared (FTIR) to understand the chemical interactions and formation of the IPN blend structure as well as to confirm successful coating with the stearic acid. X-ray diffraction (XRD) indicated the distribution of INH, while differential scanning calorimetry (DSC) was used for investigating the thermal stability of the IPN blend matrices. Scanning electron microscopy (SEM) was used to distinguish between the morphologies of coated and uncoated microspheres. Coated microspheres were produced in the size range of 52 μm down to 502 nm with encapsulation efficiencies of 65-78%. Equilibrium swelling was studied in pH 1.2 and pH 7.4 buffer media, and the in vitro drug release showed the dependence of drug release on the cross-linking, blend ratio of the IPN matrix as well as stearic acid coating. The variations in the IPN blend ratio and cross-link density controlled the drug release up to 30 h, but the coated microspheres could reduce the burst release in the gastric stomach media, while enhancing in intestinal pH 7.4 media.
1. INTRODUCTION Hydrogels are receiving renewed interests in a variety of biomedical applications, including those of skin substitutes, adhesives, matrices for drug delivery, and scaffolds for tissue engineering.1 The hydrogels prepared from a chemical modification of polysaccharides and proteins have been the promising materials in the biomedical area due to their inherent properties such as biodegradability and biocompatibility.2-5 In particular, polysaccharide-protein combinations have generated increasing interest in recent years,1 and such materials in various configurations are the effective controlled release (CR) devices in which drug is either directly embedded as in a matrix system or a depot is surrounded by a polymeric membrane (film coating and reservoir system, etc). In the latter case, diffusion of drug through the macromolecular shell dictates the release kinetics.6 Of all the methods of drug administration, the oral route has been the most convenient and widely explored approach. However, many drugs including the emerging biotechnological peptides, proteins, and nucleic acids are not suitable for oral administration because these are subjected to massive degradation in the gastrointestinal (GI) tract, offering a low permeability through the intestinal epithelium.7 Conventional pharmaceutical forms do not always allow the proper control on drug release, which must be protected from the aggressive gastric medium to subsequently guarantee a rapid dissolution and availability.8 This has resulted in considerable efforts on developing the CR devices that are able to protect the drugs in the GI tract from degradation and enhance the drug absorption in the intestine. Such advanced oral drug delivery systems include liposomes,9 mucoadhesive patches,10,11 nanoparticles,12 adsorption enhancing agents,13 and microfabricated devices.14 Among the many biomaterials, chitosan (CS), a poly[β(1f4)D-glucopyranosamine], composed of glucosamine and a N-acetyl glucosamine units, is the second most abundant natural r 2011 American Chemical Society
biopolymer having properties such as mucoadhesivity, biocompatibility, and nontoxicity.15,16 In particular, CS has the special quality of gelling upon contact with anions, forming beads under very mild conditions. and is a weak base17 with an intrinsic pKa value of 6.5. Gelatin (gel), on the other hand, is a protein obtained by hydrolysis of naturally occurring collagen, a wellknown fiber protein within the most extracellular matrices. Carboxyl groups on its backbone could form hydrogen bonds with CS to generate a well-mixed blend,18 mainly because gel is soluble in water and reacts to give cross-linked insoluble gels.1 In the present investigation, chitosan and gelatin blend interpenetrating polymer network (IPN) matrices in the form of microspheres are produced as CR devices for isoniazid (INH), an antituberculosis drug. The microspheres were coated with stearic acid to reduce the burst release of INH in acidic stomach, since the drug has a short half-life of 1-4 h, depending on the rate of metabolism; INH over dosage produces signs and symptoms within 30 min to 3 h after ingestion. Nausea, vomiting, dizziness, slurring of speech, blurring of vision, and visual hallucinations (including bright colors and strange designs) are among the early manifestations. Severe metabolic acidosis, acetonuria, and hyperglycemia are typical laboratory findings. It has a pronounced absorption from all three sections of the small intestine.19 Hence, it is important to develop its CR formulations to release the drug in the small intestine by reducing its burst effect in the gastric medium.20 Even though several studies have been devoted earlier on CS and gel blends as CR devices21-23 in addition to their applications as sponges24,25 and scaffolds,26,27 yet their practical applications have been hampered due to the sudden burst release in the Received: December 11, 2010 Accepted: February 14, 2011 Revised: February 8, 2011 Published: March 08, 2011 4504
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Industrial & Engineering Chemistry Research stomach,22 which prompts further modification of these devices to develop improved oral dosage formulations with a view to increase the release time in the intestine by protecting the drug in the acidic GI tract. In this study, we describe a method of preparing a pH-sensitive, gastroprotected CR system using CS/gel IPN blends that are cross-linked with glutaraldehyde to achieve the slow release of INH. The novelty of this work lies in developing a coating technique to reduce the burst effect in the stomach, while at the same time releasing INH in a controlled manner up to 30 h in the intestine. The drug-loaded microspheres have been characterized by a variety of techniques, and their in vitro release studies were investigated in acidic (pH = 1.2) as well as alkaline (pH = 7.4) media.
2. MATERIALS AND METHODS 2.1. Materials. Isoniazid was purchased from Loba Chemicals,
Mumbai, India. Chitosan (medium MW = 750 000) was purchased from Sigma Aldrich, Mumbai, India, and gelatin was purchased from s.d. fine chemicals, Mumbai, India. Analytical reagent grade 25% (v/v) glutaraldehyde (GA) aqueous solution, petroleum ether, and liquid paraffin oil were all purchased from s.d. fine chemicals. Span-80 was purchased from Loba Chemicals. Stearic acid (MW = 284.5) was purchased from Himedia, Mumbai, India. Water used was of high purity grade, double distilled, and deionized. 2.2. Preparation of IPN Blend Microspheres. IPN blend microspheres were prepared by using different ratios of CS and gel by varying the INH concentration as well as cross-linking agent, glutaraldehyde (GA), by emulsion cross-linking method.28 Briefly, 2 wt % CS solution was prepared by dissolving in 2% (w/v) acetic acid in double-distilled deionized water and stirring it continuously until a homogeneous solution is attained. Gel was then dispersed in CS solution and stirred overnight to obtain a homogeneous solution. INH was then dissolved in the above polymer blend solution, to which light liquid paraffin (100 g, w/w) containing 2% (w/w) span-80 was added slowly under constant stirring at 700 rpm speed for about 15 min. To this water in oil (w/o) emulsion, GA as a cross-linking agent containing 0.5 mL of 1 N HCl was added slowly with continuous stirring for 4 h. The hardened microspheres were separated by filtration and washed repeatedly with n-hexane to remove the light liquid paraffin oil. The microspheres were further washed with 0.1 M glycine solution and water to remove the unreacted GA. Brady’s test was performed to find any unreacted GA, but the test was negative, showing the absence of unreacted GA.29 Solid microspheres obtained were vacuum-dried at 40 C for 24 h and stored in a desiccator until further use. In all, 10 uncoated formulations were prepared per the formulation codes assigned in Table1. 2.3. Coating of IPN Blend Microspheres. The method of preparation of coated IPN blend microspheres is the same as described above for the preparation of IPN blend microspheres. To coat the microspheres, after adding GA slowly, 20 mL of 5% (w/v) stearic acid in ethanol was added to the formed microspheres and stirred for 4 h. The hardened microspheres were separated by filtration, washed repeatedly with n-hexane to remove light liquid paraffin oil. Again the microspheres were washed with 0.1 M glycine solution followed by water to remove the unreacted GA. Further, Brady’s test was performed to test for any unreacted GA, which was not detected. Solid microspheres obtained were vacuum-dried at 40 C for 24 h and stored in a desiccator until further use. Coating was done for all 10 formulations per the formulation codes given in Table 1.
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Table 1. Formulation Parameters codes
CS:gel ratio
INH (%, w/w)
GA (mL)
F1
90:10
20
5
F2
80:20
20
5
F3
70:30
20
5
F4
90:10
40
5
F5
90:10
60
5
F6 F7
90:10 90:10
20 20
2.5 7.5
F8
100:0
20
5
F9
60:40
20
5
F10
50:50
20
5
2.4. Drug Content. Estimation of drug concentration from the coated and uncoated microspheres was done per the method described before.30 Microspheres of known weight (10 mg) were ground to get the powder using an agate mortar, extracted with 50 mL of distilled water and sonicated for 60 min (UP 400s, Dr. Hielscher, GmBH, Germany). The solution was centrifuged (Jouan, MR23i, Nantes, France) to remove polymeric debris and washed twice to extract the drug completely. The clear solution was analyzed using UV spectrophotometer (Secomam, Anthelie, France) at λmax = 263 nm. The percent drug loading and percent encapsulation efficiency (EE) were calculated as weight of drug in microspheres 100 % drug loading ¼ weight of microspheres
ð1Þ actual drug loading 100 % encapsulation efficiency ¼ theoretical drug loading
ð2Þ 2.5. Swelling Experiments. Equilibrium swelling of all the
coated and uncoated microspheres was determined gravimetrically by measuring the extent of swelling of the microspheres in buffer media of pH 1.2 and pH 7.4. To ensure complete equilibration, samples were allowed to swell for 24 h and the excess surface-adhered liquid droplets were removed by blotting with a soft tissue paper. The swollen microspheres were weighed to an accuracy of (0.01 mg on an electronic microbalance (Mettler, model AT120, Greifensee, Switzerland). The uncoated and coated IPN blend microspheres were dried in an oven at 60 C for 5 h until no weight gain of the dried samples was observed. The percent equilibrium swelling was calculated as Ws - Wd 100 ð3Þ % swelling ¼ Wd where Ws is the weight of the swollen microspheres and Wd is the weight of the dry microspheres. Experiments were performed in triplicate, but average values within (3% standard errors were considered in data analysis and display. The percent swelling results are included in Table 2. 2.6. In Vitro Release Experiments. Drug release from the coated as well as uncoated IPN microspheres containing different drug loading, IPN composition, and extent of cross-linking were investigated in pH 1.2 for the initial 2 h, followed by the release in phosphate buffer of pH 7.4 until the completion of 4505
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Table 2. Results of Encapsulation Efficiency (EE) and Percent Swelling (S) for Uncoated and Coated Formulations in Acidic and Basic Media and Volume Mean Diameter (Vd) for Coated Formulations % S in uncoated
% S in coated Vd (μm)
codes
% EE uncoated
pH = 1.2
pH = 7.4
% EE coated
pH = 1.2
pH = 7.4
F1
59
82
63
68
45
85
52
F2
61
78
57
71
42
83
35
F3 F4
65 64
72 80
55 61
73 70
40 44
80 86
12 54
F5
70
81
59
78
46
87
60
F6
56
90
73
65
55
92
62
F7
69
60
48
72
36
75
25
F8
57
85
68
66
50
82
70
F9
66
65
51
74
35
75
0.1
F10
69
62
50
78
34
73
502 nm
the dissolution process. These experiments were performed in triplicate in a tablet dissolution tester (LabIndia, Disotest, Mumbai, India) equipped with eight baskets (glass jars) at the stirring speed of 100 rpm. A weighed quantity of each sample was placed in 500 mL of dissolution medium maintained at 37 C. At regular intervals of time, sample aliquots were withdrawn and analyzed using UV spectrophotometer (Secomam, Anthelie, France) at the fixed λmax = 263 nm. 2.7. Release Kinetics Studies. To describe the kinetics of drug release from the coated formulations, various empirical equations such as zero-order rate equation, which describes the systems where the release rate is independent of the concentration of dissolved species; the first-order equation that describes the release from systems where the dissolution rate is dependent on the concentration of dissolved species; the Higuchi square root equation that describes the release from systems where the solid drug is dispersed in an insoluble matrix and the rate of drug release is related to the rate of drug diffusion; and the HixsonCrowell cube root equation that describes the release rate from a system where there is a change in surface area and the diameter of the particles.31 The applicability of all of these equations was tested for the coated formulations that are given as follows. zero-order equation: Q ¼ Q 0 - K0 t
ð4Þ
where Q is the amount of drug remaining at time, t, Q0 is the amount of drug remaining at t = 0, and K0 is the zero-order release constant; first-order equation: ln Q ¼ ln Q0 K1 t
ð5Þ
where K1 is the first-order release constant; Higuchi square root equation: Mt ¼ KH t 1=2
ð6Þ
where Mt is the amount of drug released at t and KH is Higuchi rate constant; Hixson-Crowell cube root equation: Q 1=3 ¼ Q0 1=3 - Kc t where Kc is the cube root law release constant.
ð7Þ
All of the above equations have been tested using the release data to estimate the release kinetics parameters by the leastsquares analysis at 95% confidence limit. In addition, the following empirical equation is used as before32,33 to estimate the n and k values. Mt ¼ kt n M¥
ð8Þ
Here, Mt/M¥ is fractional drug release at t; k is a kinetic parameter that represents the drug-polymer interaction, and n is an empirical parameter characterizing the nature of the release mechanism. The estimated values of n and k for all formulations are given in Table 3. It is, however, understood that if the values of n = 0.5, drug diffuses and releases out of the polymer matrix following the Fickian diffusion. If n > 0.5, anomalous or nonFickian transport is operative. If n = 1, non-Fickian or more commonly called case II transport is present. For values of n varying between 0.5 and 1.0, transport is anomalous type.32,33
3. CHARACTERIZATION 3.1. Fourier Transform Infrared Spectral Measurements. Fourier transform infrared (FTIR) spectra were obtained using Nicolet (Model Impact 410, Milwaukee, WI, USA) instrument to confirm the formation of IPN structure as well as to confirm the coating and to find any chemical interactions of INH with the polymer. FTIR spectra of the plain CS, plain gel, placebo microspheres, uncoated drug-loaded microspheres, coated drugloaded microspheres, and pristine INH were all obtained under identical conditions. Samples were ground with KBr, and pellets were prepared by applying a hydraulic pressure of 600 kg/cm2. Spectral scanning was done in the range of 500-4000 cm-1. 3.2. Differential Scanning Calorimetric Study. Differential scanning calorimetry (DSC) and thermogravimetric analysis (DSC-Q20, TA Instruments-Waters, Schaumburg, IL, USA) was performed on plain CS, plain gel, uncoated placebo microspheres, and uncoated and coated isoniazid-loaded microspheres as well as pristine isoniazid. Samples were heated from 25 to 400 C at the rate of 10 C/min in a nitrogen atmosphere. 3.3. X-ray Diffraction Study. Crystallinity of INH after encapsulation was evaluated by X-ray diffraction (XRD) measurements recorded for uncoated placebo microspheres, coated isoniazid-loaded microspheres, and pristine isoniazid using X-ray 4506
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Table 3. Kinetics Parameters with Correlation Coefficients of Different Equations codes F1 F2 F3
Higuchi square root rate constant (eq 5; pH = 1.2]
k
n
r
0.15
0.59
0.942
0.117
0.70
0.922
0.085
0.91
0.989
zero-order rate constant (eq 3; pH = 7.4]
2.916
1.3193
R = 0.9928
R = 0.9074
2.3353
1.1649
R = 0.979
R = 0.9647
1.6564
0.8895
R = 0.9119
R = 0.9576
F4
4.1374
1.7439
0.199
0.56
0.918
F5
R = 0.9864 6.1756
R = 0.8651 2.4596
0.300
0.50
0.952
R = 0.9676
R = 0.817
5.4157
2.3228
0.257
0.43
0.904
R = 0.982
R = 0.8799
1.6469
0.7635
0.111
0.53
0.773
R = 0.9595
R = 0.907
F8
6.9988
3.3298
0.291
0.47
0.987
F9
R = 0.9878 1.3116
R = 0.9406 0.7333
0.0373
1.49
0.945
R = 0.8553
R = 0.9069
0.9683
0.5333
0.0353
1.46
0.910
R = 0.902
R = 0.9236
F6 F7
F10
Figure 1. FTIR of chitosan, gelatin, placebo microspheres, drug, placebo, uncoated microspheres, and coated microspheres.
diffractometer (x-Pert, Philips, Sheffield, U.K.). Scanning was done up to 2θ = 80. 3.4. Scanning Electron Microscopic Study. Scanning electron microscope (SEM) images were taken using a JEOL model JSM-840A, Japan instrument (available at Shivaji University, Kolhapur, India). Microspheres were sputtered with gold to make them conducting and placed on a copper stub. Thickness of the gold layer accomplished by gold sputtering was about 10 nm. 3.5. Particle Size Measurements. Particle size and size distributions were measured using a mastersizer (Malvern, model MS-2000, Malvern, U.K.). Particle size data of different coated formulations are also included in Table 2.
4. RESULTS AND DISCUSSION 4.1. Fourier Transform Infrared Spectral Study. FTIR spectra (see Figure 1) of the plain CS, plain gel, and uncoated placebo microspheres were taken to prove the formation of IPN structure. In the case of CS, a broad band observed at 3427 cm-1 is attributed to O-H stretching vibrations, while the bands at 2922 and 2810 cm-1 represent the presence of C-H aliphatic stretching vibrations. Three bands appearing at 1651, 1590, and 1379 cm-1 are assigned to amide-I, amide-II, and amide-III, respectively. The CS is characterized by its saccharide structure at 899 and 1154 cm-1. In the case of gel, a characteristic band due 4507
dx.doi.org/10.1021/ie102479m |Ind. Eng. Chem. Res. 2011, 50, 4504–4514
Industrial & Engineering Chemistry Research Scheme 1. Formation of Hybrid IPN from CS and Gel and Formation of Stearic Acid-Coated Hybrid IPN Microsphere
to N-H stretching is observed at 3442 cm-1. The N-H bending vibration is observed at 1535 cm-1, but aliphatic C-H bending vibrations are observed at 1454 and 1405 cm-1. The band appearing at 1638 cm-1 indicates amide-I band, while bands at 1321 and 1237 cm-1 indicate the C-N bond stretching vibrations. In the case of placebo microspheres, all of the characteristic bands of both CS and gel are observed in addition to new bands at 1654 and 1563 cm-1. These peaks indicate C-N stretching vibration of the imine group of Schiff base formed via co-crosslinking reactions between CS, gel, and glutaraldehyde.22 Thus, FTIR data confirm successful cross-linking of both CS and GEL to form IPN blend structure in the presence of GA as shown in Scheme 1. FTIR spectra of the drug, placebo, and uncoated and coated microspheres were taken to investigate stearic acid coating and chemical stability of INH after encapsulation. In the case of pristine INH, a band at 3300 cm-1 is due to N-H stretching vibrations, while that at 3180 cm-1 is for NH2 stretching vibrations. The bands at 3061, 3013, 2949, and 2854 cm-1 are due to aromatic and aliphatic C-H stretching vibrations. The band at 1666 cm-1 is assigned to carbonyl of amide group stretching vibrations. Another band at 1221 cm-1 is due to C-N stretching vibrations, while a band at 1556 cm-1 is attributed to N-H bending vibrations. In case of drug-loaded microspheres, all the bands that are observed for INH have also appeared, indicating no chemical interaction of INH with the IPN matrix even after encapsulation. An extra carboxylate anion band at 1545 cm-1 is observed in the coated microspheres, which is absent in the uncoated microspheres, indicating electrostatic interactions between chitosan and stearic acid, which confirms the successful coating of the microspheres. 4.2. Differential Scanning Calorimetric Studies. DSC was used to study thermal transitions during heating cycles under inert atmosphere. Typical DSC thermograms of CS, gel, INH,
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placebo microspheres and drug-loaded coated microspheres are displayed in Figure 2. In the case of pure INH, a sharp peak at 172 C is observed, indicating its melting point. The thermogram of the placebo microspheres shows a peak at 80 C due to endothermic transition as a result of loss of moisture from CS and gel IPN matrix. A peak at 280 C is observed due to exothermic transition of the IPN matrix as a result of decomposition of CS as well as gel. Also, drug-loaded microspheres have shown patterns similar to that of placebo, but no peaks are observed at 172 C, indicating the amorphous nature of the polymer even after encapsulation of INH into IPN matrix. 4.3. X-ray Diffraction Study. X-ray diffractograms of the coated drug-loaded microspheres, placebo microspheres, and pristine INH presented in Figure 3 are used to investigate the drug’s polymorphism after encapsulation. The diffraction pattern of INH shows intense peaks at 2θ = 14, 16, and 20 that are characteristics of its crystalline nature. However, these peaks have disappeared and broadened in the INH-loaded microspheres. The XRD peak depends on crystal size, and in the present study, for drug-loaded formulation, the characteristic peak of INH has merged with the polymer-inducing amorphous phase. However, it is difficult to study drug’s crystallinity at the detection limit of the crystal size in the case of drug-loaded microspheres. XRD data suggest the molecular dispersion of INH in the polymer matrix. 4.4. Scanning Electron Microscopic Study. SEM images of the microspheres taken at 500, 1500, 1900, 4000, and 5000 magnifications shown in Figure 4 are spherical with the smooth surfaces. In a few cases, however, some surface-adhered drug particles are seen. In Figure 4A,B, the microspheres have wrinkled surfaces due to coating of stearic acid onto the surface of microspheres.34 Parts C and D of Figure 4 (F9) show much smaller particles compared to those in A and B because of higher gel concentration of the IPN matrix, suggesting its rigid nature. 4.5. Particle Size. The results of mean particle size are presented in Table 2, while the size distribution curve for typical formulation F3 containing 30 wt % gel, 70 wt % CS with 20 wt % INH, and 5 mL of GA, are displayed in Figure 5. Identical to the results of % EE and the percent swelling, the size of the microspheres depends on the amount of drug loaded in the IPN matrix, percent gel content, and extent of cross-linking of the matrix. The size of the microspheres range between 52 μm and 500 nm, but with increasing concentration of gel in the IPN matrix, the size of the microspheres decrease drastically from 52 μm to 502 nm. The particle size of F2 (20% (w/w), gel) is lower than that of F1 (10% (w/w), gel), and similar trends are observed with F3, F9, and F10 formulations. The reason for such a variation is higher concentration of gel in the blend IPN matrix, thereby increasing the rigidity of the matrix. The size of the coated microspheres is less than those of the uncoated microspheres, due to electrostatic interactions between the negatively charged stearic acid and the positively charged CS as well as gel, thereby leading to shrinkage of the size of microspheres. Particle size shows a dependence on the extent of drug loading. For instance, formulations containing 40 wt % (F4) and 60 wt % drug (F5) exhibit higher sizes than those containing 20 wt % drug (F1) due to the interstitial accommodation of more drug particles during the formulation process. The particle size decreases with increasing extent of cross-linking, as seen in case of F1, whose size is 1, indicating super-case II transport. However, for F6 and F8, the n values are
