ARTICLE pubs.acs.org/IECR
Novel Semi-interpenetrating Microspheres of Dextran-grafted-Acrylamide and Poly(Vinyl Alcohol) for Controlled Release of Abacavir Sulfate Anita G. Sullad,† Lata S. Manjeshwar,*,† and Tejraj M. Aminabhavi‡ † ‡
Department of Chemistry, Karnatak University, Dharwad 580 003, India CSIR Emeritus Scientist, SET’s College of Pharmacy, Dharwad 580 002, India ABSTRACT: A novel drug delivery system using semi-interpenetrating (semi-IPN) microspheres of dextran-grafted-acrylamide (Dex-g-AAm) and poly(vinyl alcohol) (PVA) was prepared in the size range of 80100 μm by emulsion cross-linking for investigating controlled release (CR) of an anti-HIV agent, abacavir sulfate. The graft copolymer was confirmed by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The microspheres were characterized for morphology, swelling, and in vitro release of abacavir sulfate in pH 1.2 and 7.4 buffer media to display the effect of drug release in acidic and alkaline conditions. The kinetics of in vitro release was analyzed using the empirical equations to understand the nature of release mechanism.
1. INTRODUCTION Synthetic and natural polymers or their combinations have been widely used in biomedical area.13 Many times, properties of pure synthetic polymers and those of pure natural polymers alone are inadequate to develop materials having a good combination of chemical, mechanical, thermal or biological performance. Therefore, modification of natural polymers such as dextran (Dex),4 starch,5 guar gum6 and chitosan7 have been attempted with the synthetic versions. Among various such methods employed, graft copolymerization has been widely used to modify the chemical and physical properties of natural polymers.8,9 In graft copolymerization, the guest monomer benefits the host polymer with some novel and desired properties in which the resultant copolymer gains the characteristic properties that are useful for controlled release (CR) applications. Graft copolymerization of vinyl monomers onto natural polymers introduce the desired properties as these have enlarged their potential applications in biomedical area.10,11 However, the grafting of natural polymers using ceric ammonium nitrate is the most extensively investigated approach.12,13 Dex, a glucose homopolysaccharide, features substantial number of consecutive α-(1f6) linkages in the backbone, usually >50% of the total linkages. These α-D -glucans possess side chains stemming from α-(1f2), α-(1f3), or α-(1f4) branch linkages. Dex has been extensively used in biomedical area as a drug carrier because of its excellent biocompatibility,14,15 but its uncontrolled rate of hydration and low mechanical strength, limits its long-term application. Thus, there is a need to modify Dex to be useful as a drug delivery device. Earlier, Rokhade16 et al., developed semi-IPN microspheres of acrylamide grafted onto Dex and chitosan used in the CR of acyclovir to observe that % cumulative release rates depend on the nature of the polymeric carrier. Cascone et al.,17 demonstrated the release of dexamethasone from PLGA nanoparticles encapsulated into dextran/poly(vinyl alcohol) (Dex/PVA) hydrogel matrix. In our earlier efforts, a number of interpenetrating network (IPN)-based formulations have been developed as CR devices r 2011 American Chemical Society
for the CR of a variety of drugs.18,19 In continuation of these studies, we now report here the synthetic protocols for the preparation of semi-IPN microspheres of dextran-grafted-acrylamide (Dex-g-AAm) and PVA as the effective CR device for abacavir sulfate, an antidepressant drug of the antiviral/reverse transcriptase inhibitor class. Chemically, the drug is (1S,4R)-4-[2amino-6(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1methanol sulfate (see Figure 1), which prevents HIV. The drug has a plasma half-life of 1.45 h, which calls for developing a CR formulation. Therefore, in this study, semi-IPN microspheres were prepared by the water-in-oil (w/o) emulsion cross-linking method and were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) techniques. In vitro release studies have been performed to understand their in vitro release kinetics in pH 1.2 and 7.4 buffer media. Release data of this study have been analyzed using empirical kinetic equations.
2. EXPERIMENTAL SECTION 2.1. Materials. PVA (M w = 125 000) of degree of hydrolysis 8689%, acrylamide, cerric ammonium nitrate, analytical reagent grade glutaraldehyde (GA) solution of 25% (v/v), Tween80, petroleum ether, and liquid paraffin oil were all purchased from s.d. fine Chemicals, Mumbai, India. Dextran (M w = 10 000) was purchased from Hi-media Chemicals Pvt. Ltd., Mumbai, India. Abacavir sulfate was received as a gift sample from the local drug company (a proprietary item). A high purity grade deionized water was used. 2.2. Preparation of Dex-g-AAm. Graft copolymerization was carried out by free-radical polymerization.13 In brief, 2 g of Dex was dissolved in 70 mL of water, which was allowed to hydrate and was dissolved under constant stirring for overnight in Received: March 30, 2011 Accepted: August 24, 2011 Revised: July 16, 2011 Published: August 24, 2011 11778
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250 mL round-bottom flask. Then, 0.12 mol of acrylamide dissolved in 20 mL of water was added to Dex solution and mixed under moderate stirring for 1 h. To this solution was added 5 mmol cerric ammonium nitrate and polymerization was carried out at 60 °C under continuous purging of nitrogen gas for 6 h in a water bath with constant stirring. After completing the polymerization reaction, the polymer was cooled under running tap water and poured into excess acetone to induce precipitation. The solid graft copolymer thus obtained was washed several times with methanol/water (80:20) mixture to remove the homopolymer and vacuum-dried at 40 °C to constant weight. Schematic representation of the synthesis of Dex-g-AAm is shown in Figure 1. The % grafting efficiency was calculated as W1 W0 100 ð1Þ % grafting efficiency ¼ W2
solution containing 0.5 mL of 0.1 N HCl were added and the mixture was stirred for 3 h. The hardened microspheres were filtered and washed with petroleum ether and water to remove the unreacted GA along with the adhered Tween-80. Solid microspheres were vacuum-dried at 40 °C for 24 h and stored in a desiccator until further use. The formulations with different compositions are listed in Table 1. 2.4. Fourier Transform Infrared (FTIR) Spectroscopy. The formation of (Dex-g-AAm) copolymer and chemical stability of the drug in the polymer matrix after encapsulation were examined by FTIR. The FTIR spectra of PVA, Dex, Dex-g-AAm, placebo PVA-(Dex-g-AAm) microspheres, abacavir sulfate-loaded PVA-(Dex-g-AAm) microspheres, and pristine abacavir sulfate were obtained using Nicolet spectrophotometer (Impact 410; Milwaukee, WI) in the wavelength region of 5004000 cm1. Microspheres were crushed to make KBr pellets under a hydraulic pressure of 600 kg/cm2. 2.5. Differential Scanning Calorimetry (DSC). Differential scanning calorimetry experiments were performed on pristine PVA, pristine Dex, Dex-g-AAm, placebo PVA-(Dex-g-AAm) microspheres, abacavir sulfate-loaded PVA-(Dex-g-AAm) microspheres and pristine abacavir sulfate using Q20 DSC apparatus (TA Instruments-Waters, U.S.A.). Samples were heated from 25° to 400 °C at the heating rate of 10 °C/min in a nitrogen atmosphere. 2.6. X-ray Diffraction (XRD). Crystallinity of abacavir sulfate after encapsulation was evaluated by XRD recorded for pristine PVA, placebo and abacavir sulfate-loaded PVA-(Dex-g-AAm) microspheres as well as pristine abacavir sulfate using X’Pert (Philips, Guildford, Surrey, U.K.) X-ray diffractometer. Scanning was done at ambient temperature (30 °C) by varying the angle, 2θ up to 50°. 2.7. Scanning Electron Microscopy (SEM). Samples of the microspheres were taken on a copper stub and sputtered with gold for 2 min. The gold-coated (10 nm thick) microspheres were mounted on SEM instrument (JEOL model JSM-840A, Tokyo, Japan), and images were taken at 1500 magnifications. 2.8. Swelling Studies. Equilibrium swelling of the microspheres was measured by determining the weights of the samples to monitor the extent of swelling in pH 7.4 buffer medium. Microspheres were allowed to swell completely for up to 24 h to attain equilibrium saturation at the physiological temperature (37 °C). Adhered liquid droplets on the surface of microspheres were removed by blotting with soft filter papers and swollen microspheres were weighed on an electronic balance (model AT20, Mettler, Greifensee, Switzerland). Microspheres were dried in an oven at 40 °C for about 5 h until there was no change in the dry mass of the samples. The % equilibrium swelling was calculated as before.20
where W0, W1, and W2 are the weights of Dex, graft copolymer, and monomer, respectively. 2.3. Preparation of PVA-(Dex-g-AAm) Microspheres. Microspheres of PVA-(Dex-g-AAm) polymer were prepared by water-in-oil (w/o) emulsion cross-linking method.19 The 20 mL of 2% (w/v) polymer solution was prepared by dissolving varying amounts of PVA and Dex-g-AAm in double distilled deionized water. Different quantities of abacavir sulfate were dissolved in polymer blend solution and emulsified slowly into light liquid paraffin (100 g, w/w) containing 1% (w/w) Tween-80 under constant stirring at 400 rpm using a Eurostar high-speed stirrer (IKA Labortechnik, Staufen, Germany) for about 15 min. To this w/o emulsion, three concentrations viz., 2.5, 5, or 10 mL of GA
Figure 1. Schematic representation of the formation of dextran-gacrylamide and chemical structure of abacavir sulfate.
Table 1. Formulation Parameters, % Equilibrium Swelling, Empirical Parameters, n, k, and Correlation Coefficient (r2) of eq 6 from eq 6 formulation codes
PVA (% w/w)
Dex-g-AAm (% w/w)
abacavir sulfate loading (%)
GA (mL)
% equilibrium swelling
n
k 102
r2
F1
90
10
10
2.5
186
0.77
1.138
0.969
F2
90
10
10
5
100
0.52
4.691
0.925
F3
90
10
10
10
58
0.74
1.261
0.977
F4 F5
80 70
20 30
20 30
5 5
151 205
0.54 0.41
3.736 8.652
0.962 0.942
FP
100
-
10
5
93
0.49
6.087
0.953
11779
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2.9. In Vitro Drug Release. Drug release from the blend matrix follows several types of mechanisms: namely, (a) release from the surface of microspheres, (b) diffusion through the swollen rubbery blend matrix, and (c) release due to polymer erosion. In case of release from the surface, the adsorbed drug particles will instantaneously dissolve in the presence of the release medium. Thus, drug encapsulated in the surface layer of the microspheres follows surface erosion mechanism, leading to initial burst effect. To understand the drug release from the microspheres in the stomach and intestinal conditions, in vitro release experiments from the microspheres with different polymer blend compositions and different extent of cross-linking were investigated in 1.2 pH buffer media for the initial 2 h, followed by phosphate buffer at pH 7.4, until the completion of dissolution process. These experiments were performed in triplicate using the tablet dissolution tester (LabIndia, Mumbai, India) equipped with eight baskets (glass jars) at the stirring speed of 100 rpm. Weighed quantity of each sample was placed in 500 mL of the dissolution medium maintained at 37 °C. At regular intervals of time, 5 mL of sample aliquots were withdrawn and analyzed by UV spectrophotometer (Secomam, Anthelie, France) at the fixed λmax of 288 nm. The utilized solvent media was replenished by adding 5 mL of fresh solvent. Triplicate data were collected, but the cumulative release curves were drawn through average points, giving standard deviations of (3% in all the formulations. 2.10. Empirical Analysis of In Vitro Release Data. To ascertain the kinetics of drug release, following empirical equations21 were used and release kinetics parameters were evaluated. The zero-order equation
Q ¼ Q 0 K0 t
ð2Þ
where Q is the amount of drug remaining at time, t; Q0 is the amount of drug remaining at t = 0, and K0 is zero-order release constant. First-order equation ln Q ¼ ln Q0 K1 t
ð3Þ
where K1 is the first-order release constant. Higuchi square root equation Mt ¼ KH t 1=2
ð4Þ
where Mt is the amount of drug released at time, t and KH is Higuchi rate constant. HixsonCrowell cube root equation Q 1=3 ¼ Q0 1=3 Kc t
ð5Þ
where Kc is cube root law release constant. Cumulative release data were also analyzed using22 Mt =M∞ ¼ kt n
ð6Þ
Here, Mt/M∞ represents fractional drug release at time, t, k is a kinetic parameter characterizing drug-polymer interaction, and n is an empirical parameter characterizing the release mechanism. Dissolution profiles of all the formulations have been tested using the least-squares method at 95% confidence limit using eqs 26 in both the pH media of 1.2 and 7.4. For microspheres, a value of n = 0.43 indicates Fickian or diffusion mechanism. If n = 0.85, non-Fickian or more commonly called Case II transport occurs. If the values of n vary between 0.43 and 0.85, then transport follows anomalous trend.23
Figure 2. FTIR spectra of (a) poly(vinyl alcohol), (b) dextran, (c) dextran-g-acrylamide, (d) placebo poly(vinyl alcohol)dextran-g-acrylamide microspheres, (e) abacavir sulfate-loaded poly(vinyl alcohol) dextran-g-acrylamide microspheres, and (f) pristine abacavir sulfate.
3. RESULTS AND DISCUSSION Graft copolymerization of dextran with AAm was achieved by Ce(IV) catalyzed free radical polymerization. The chelate complex formed with the OH group of Dex decomposes to generate free radical site, thereby facilitating grafting to occur at the active site of dextran with the incoming AAm monomer (see Figure 1). The grafting efficiency as calculated from eq 1 was found to be 68%. 3.1. Fourier Transform Infrared Spectra. FTIR spectra of pristine Dex and Dex-g-AAm confirmed the grafting reaction as shown in Figure 2 for (a) pristine PVA, (b) Dex, and (c) Dex-gAAm. In case of PVA, a broad band observed at 3336 cm1 is attributed to OH stretching vibrations. Two bands observed at 2937 and 2860 cm1 represent the presence of CH aliphatic stretching vibrations, while for Dex, two characteristic peaks appear at 3422 and 1344 cm1, respectively, because of the OH stretching and bending vibrations, while the bands appearing at 2924 and 2855 cm1 are due to CH stretching vibrations. The peak at 1411 cm1 is due to CH bending vibrations. Appearance of a new sharp band at 1743 cm1 of Dex-g-AAm due to carbonyl stretching along with other bands confirms the formation of graft copolymer. FTIR spectra of (d) placebo PVA-(Dex-g-AAm) microspheres, (e) abacavir sulfate-loaded PVA-(Dex-g-AAm) microspheres, and (f) pristine abacavir sulfate are shown in Figure 2. In case of pristine abacavir sulfate, primary and secondary NH stretching peak is observed at 3369 cm1. However, sharp peaks observed at 1674 cm1 and 1643 cm1 are due to CdN and CN stretchings, while the bands at 3180 and 2924 cm1 are for aromatic and aliphatic CH stretching vibrations, respectively. In case of placebo microspheres, a broad band at 3423 cm1 is attributed to OH bond stretching vibrations, while those at 2924 cm1 and 2855 cm1 are due to CH aliphatic stretching vibrations. On the other hand, a band at 1733 cm1 11780
dx.doi.org/10.1021/ie2006438 |Ind. Eng. Chem. Res. 2011, 50, 11778–11784
Industrial & Engineering Chemistry Research
Figure 3. DSC thermograms of (a) poly(vinyl alcohol), (b) dextran, (c) dextran-g-acrylamide, (d) placebo poly(vinyl alcohol)dextran-g-acrylamide microspheres, (e) abacavir sulfate-loaded poly(vinyl alcohol) dextran-g-acrylamide microspheres, and (f) pristine abacavir sulfate.
corresponds to carbonyl bond stretching vibrations. In addition, a band at 1019 cm1 is attributed to the presence of an acetal group formed between the reaction of GA and hydroxyl groups of PVA. In case of FTIR spectrum of abacavir sulfate-loaded microspheres, the NH stretching peak of abacavir sulfate has merged with the OH stretching vibrations of the polymer matrix. This is further confirmed by the shifting of OH peak from lower to higher frequency region. A characteristic peak of abacavir sulfate observed at 1631 cm1 in abacavir sulfate-loaded microspheres confirms no interactions between drug and polymer matrix, suggesting that abacavir sulfate is chemically stable in the polymer matrix. 3.2. Differential Scanning Calorimetric Studies. DSC also confirmed the formation of graft copolymers. DSC curves of (a) PVA, (b) Dex, and (c) Dex-g-AAm copolymer are displayed in Figure 3. DSC of PVA showed endothermic transition at 70 °C, while Dex showed endothermic peaks at 65° and 242 °C because of polymeric transitions, while the peak at 312 °C is due to thermal decomposition of the polymer. The graft copolymer exhibits endothermic transitions at 64, 234, and 289 °C, but the peak at 377 °C is due to thermal decomposition of Dex-g-AAm. The new endothermic transition at 289 °C for graft copolymer is due to enhanced interaction between carbonyl groups of the grafted copolymer and hydroxyl groups of Dex, thus confirming the grafting of acrylamide onto Dex. From the DSC thermograms of Dex and Dex-g-AAm, it is evident that the graft copolymer is thermally more stable than that of Dex. Figure 3 also shows DSC spectra of (d) placebo PVA-(Dex-g-AAm) microspheres, (e) abacavir sulfate-loaded PVA-(Dex-g-AAm) microspheres, and (f) pristine abacavir sulfate. The pristine abacavir sulfate shows an endothermic peak at 228 °C, while the placebo microspheres show a broad peak at 65 °C due to endothermic transition of the polymer. The abacavir sulfate-loaded microspheres have also shown similar
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Figure 4. XRD spectra of (a) poly(vinyl alcohol), (b) placebo poly(vinyl alcohol)dextran-g-acrylamide microspheres, (c) abacavir sulfate-loaded poly(vinyl alcohol)dextran-g-acrylamide microspheres, and (d) pristine abacavir sulfate.
curves as that of placebo, but no characteristic peaks of abacavir sulfate were observed, indicating that drug is molecularly dispersed in the polymer matrix. 3.3. X-ray Diffraction Studies. X-ray diffractograms of (a) pristine PVA, (b) placebo PVA-(Dex-g-AAm) microspheres, (c) abacavir sulfate-loaded PVA-(Dex-g-AAm) microspheres, and (d) pristine abacavir sulfate are presented in Figure 4. The XRD was used to investigate the crystallinity of abacavir sulfate in the cross-linked microspheres. For instance, XRD of pristine PVA shows intense peak around 2θ of 20° and diffraction patterns of abacavir sulfate have many peaks around 2θ of 930°, while a high intensity peak at 2θ = 21° confirms its crystalline nature. However, these peaks have disappeared in abacavir sulfate-loaded microspheres and only the peaks observed in placebo polymer are seen, confirming that drug is molecularly dispersed in the polymer matrix and no crystals were found in abacavir sulfate-loaded formulations. 3.4. Scanning Electron Microscopic Analysis. SEM images of the group of (a) PVA-(Dex-g-AAm) microspheres and (b) surface morphology of the microspheres taken at magnifications of X150 and X1500 are shown in Figure 5. The microspheres are somewhat spherical in nature having rough surfaces, exhibiting pores on the surface, which might have been created during the particle hardening stage. The size of the microspheres vary from 80 to 100 μm. 3.5. Swelling Studies. To investigate the effect of crosslinking and polymer blend ratio on the swelling of microspheres, we have measured the % equilibrium swelling of the microspheres in both 1.2 pH and 7.4 pH buffer media. These data are also included in Table 1. Swelling of the formulations in both the media was found to depend strongly on the extent of 11781
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Figure 7. Effect of polymer blend ratio on in vitro release for formulations: F2 (10% w/w dextran-g-acrylamide), F4 (20% w/w dextran-gacrylamide), F5 (30% w/w dextran-g-acrylamide), and FP (0% w/w dextran-g-acrylamide) at 37 °C.
Figure 5. SEM images of (a) abacavir sulfate-loaded poly(vinyl alcohol)dextran-g-acrylamide microspheres and (b) surface of abacavir sulfate-loaded poly(vinyl alcohol)dextran-g-acrylamide microspheres.
Figure 6. Effect of cross-linking on in vitro release for formulations: F1 (2.5 mL of GA), F2 (5 mL of GA), and F3 (10 mL of GA) at 37 °C.
cross-linking of the matrix. Equilibrium swelling decreased with increasing cross-link density. At lower cross-link density, swelling increased, because the network structure becomes loose as a result of creation of large hydrodynamic free volume, such that the polymer chains will accommodate more of solvent molecules, resulting in increased swelling. The glass transition temperature
(Tg) of the polymer has also increased with increasing cross-link density, since the glassy polymer does not have the loose macromolecular network structure, thus resulting in a low water sorption. Another important criterion on which swelling depends is polymer blend composition. For instance, the virgin PVA formulation has shown the least swelling (93%) compared to its blends with Dex-g-AAm (196%). This is because of the presence of AAm, which might have increased the hydrophilic nature of the matrix. As the Dex-g-AAm content of the blend matrix increased from 10 to 30% (as in F2, F4, and F5), swelling also increased from 100 to 205%. 3.6. In Vitro Drug Release Kinetics. 3.6.1. Effect of CrossLinking. Figure 6 displays the release profiles of microspheres cross-linked with different concentrations of GA in the matrix that contained 10% (w/w) of Dex-g-AAm and 10% (w/w) of abacavir sulfate (i.e., F1, F2, and F3 formulations). The cumulative release is
