SODIUM ALGINATE/GELATIN MICROBEADS-INTERCALATED WITH KAOLIN NANOCLAY FOR EMERGING DRUG DELIVERY IN WILSON’S DISEASE

Objective: The aim of the present study was to fabricate and evaluate the drug release studies using Sodium Alginate (SA) and Gelatin (GE) microbeads intercalated with Kaolin (KA) nanoclay for sustained release of D-Penicillamine (D-PA). Methods: Sodium alginate/gelatin/Kaolin blend microbeads were prepared by an extrusion method by using glutaraldehyde (GA) as a crosslinker. The obtained microbeads were characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and X–ray diffraction (XRD). Drug release kinetics of the microbeads was investigated in simulated intestinal fluid (pH 7.4) at 37 °C. Results: Microbeads formation was confirmed by FTIR spectroscopy. X-RD reveals that the KA should be intercalated with the drug and also it confirms the molecular level dispersion of D-Penicillamine into microbeads. Scanning Electron Microscopy (SEM) studies reveal that the beads were in spherical shape with some wrinkled depressions on the surface. The in vitro release study indicates the D-Penicillamine released in a controlled manner. The in vitro release kinetics was assessed by Korsmeyer-Peppas equation and the ‘n’ value lies in between 0.557-0.693 indicates Non-Fickian diffusion process. Conclusion: The results suggest that the developed KA intercalated microbeads are good potential drug carrier for the controlled release of D-PA. some wrinkled depressions on the surface. The in vitro release study indicates the D-PA released in a controlled manner. The in vitro release kinetics was assessed by different empirical equations and the data supported a Non-Fickian diffusion mechanism. Based on the results, Kaolin intercalated microbeads are promising material for drug carrier in emerging drug delivery systems.

Kaolin, Al2Si2O5 (OH)4 (KA) is a two-dimensional (2D) natural aluminosilicate mineral which has been used in the field of biomedical applications for centuries [14]. In fact their medicinal utilities have been discovered by many traditional civilizations (Egyptians, Assyrians, Babylonians, Indians, Chinese), Greeks, Romans and medieval Arab Muslims till the recent times [15]. Kaolinite is used in the treatment of colitis, enteritis, dysentery and diarrhoea [16]. In the last few decades, researchers have paid huge attention to use KA minerals, because it can acts as active excipient in solid and semisolid pharmaceutical dosage forms and control the efficiency of the dosage forms which can improve the drug bioavailability [17][18][19].
Sodium Alginate (SA) is an anionic biodegradable natural polymer with 1,4-β-d-mannuronic acid (M block) and α-l-guluronic acid (Gblock) residues [20]. The polymer forms a three-dimensional hydrogel which network due to the interaction between the carboxylic acid group (Gblock) and divalent counter ion (e. g Ca 2+ , Mg 2+ and Zn 2+ ) and makes egg-box structure, results a cross linked hydrogels is useful in controlled release of bioactive molecules [21,22]. SA crosslinked calcium beads could protect the acid sensitive drugs from gastric juice and consequently released from the beads in the intestine [23]. F. Martínez-Gómez et al., [24] reported that sodium alginate/polyvinyl alcohol hydrogels may be appropriate for use in the controlled release of drugs in the intestinal tract.
Gelatin (GE) is a natural protein material obtained by hydrolytic degradation of naturally occurring tropocollagen rod (a triple helical structure) which is a fundamental molecular unit of collagen [25,26]. In the recent decades it has been used in food and pharmaceutical industries due to its non-toxic, anti-carcinogenic, biocompatible, biodegradable, and also attractive starting material as a drug carrier due to its unique gelling properties [27]. Phadke et al., [28] reported that it was somewhat difficult to prepare long-lasting drug delivery vehicle because at body temperature it was dissolved very rapidly, makes a burst release of drug. In order to overcome these problems many researchers have prepared GE networks by crosslinking (Glutaraldehyde, formaldehyde and genipin), grafting and coating with other polymers. A. P. Rokhade et al., [29] reported that gelatin and NaCMC semi IPN microspheres for the effective encapsulation and controlled release of ketorolac tromethamine. Pal et al., [30] reported that gelatin-g-PAA nanoparticles are fairly suitable and function well as swelling controlled drug delivery system. D-Penicillamine (D-PA) ( fig. 1) is a biologically active aminothiol [31], which is used in the treatment of Wilson's disease and remove the excess of copper from the body, may be in the form of deep purple colour complex with D-PA by chelation process [32]. D-P is a potentially an effective chelator for some transition metals, hence it is used as a good choice for treatment of heavy metal poising [33].
A few articles have been reported the combination of SA and GE for controlled drug delivery [34][35][36][37], but as per best of our knowledge, the use of SA/GE/KA microbeads for controlled release of D-PA has not yet been investigated. In this research paper KA intercalated SA/GE microbeads were prepared. The developed beads were characterized by FTIR, SEM, EDS and X-RD. The dissolution experiments were performed to study the drug release characteristics of the microbeads and the results are presented here.

Materials
Gelatin and kaolin were purchased from Sigma-Aldrich (USA). Sodium alginate, Calcium chloride and Glutaraldehyde (GA) were purchased from Sd. Fine chemicals, Mumbai, India. D-Penicillamine a gift sample from Suven Life Sciences Ltd., Hyderabad. Water used was of high purity grade after double distillation.

Preparation procedure
Preparation of SA/GE microbeads D-PA loaded microbeads were prepared by simple ionotropic gelation method [25]. Briefly, 3% aqueous dispersion of SA and GE were prepared separately using double distilled water. Solutions of GE and SA were combined in different ratios (table 1). To this drug solution was added and stirred for 2hr by using a magnetic stirrer to get homogeneous solution. The above mixed solution was transferred drop wise into CaCl2 solution, the spherical beads formed instantly were kept for 5 min-and then transferred into 2% glutaraldehyde solution for crosslinking for 2 h. The obtained wet beads were collected by decantation, washed three times with distilled water to remove the drug attached on the bead surface, and finally were dried in air overnight and then vacuum-dried to constant weight at 40 °C.

Preparation of SA/GE-kaolin nanoclay microbeads
SA/GE blend solution was prepared by using above procedure. To this solution drug and nano kaolin clay was added in different ratios (table 1) and stirred for 2hr using magnetic stirrer. This solution was placed in sonication for 30 min to get homogeneous solution.
Then the solution was transferred drop wise into CaCl2 solution, the spherical microbeads formed instantly were kept for 5 min and then transferred into 2% glutaraldehyde solution for crosslinking for 2 h. The obtained wet beads were collected by decantation, washed three times with distilled water to remove the drug attached on the bead surface, and finally were dried in air overnight and then vacuum-dried to constant weight at 40 °C.

Intercalation kinetics
To determine the optimal time required for maximum intercalation of D-PA with KA. A known amount of D-PA (30 mg) and KA (100 mg) were mixed into 30 ml of double distilled water with continuous stirring at 37 °C. At regular intervals of time (0.5, 1, 2, 4, 8 and 14 h) the reaction mixture was filtered and concentration of D-PA was analyzed using UV spectrophotometer at fixed λ-max value of 215.2 nm.

Effect of pH
The experiments were performed to estimate the most favourable pH for

Fourier transform infrared (FTIR) spectral analysis
FTIR (Bomem MB-3000 Make: Canada) with Horizon MBTM FTIR software was used to record the spectrum of pure SA, pure GE, pristine drug, placebo SA/GE microbeads, drug loaded SA/GE microbeads and drug loaded SA/GE/KA microbeads to find out the possible chemical interactions between polymer matrix, kaolin and drug. The finely powdered and dried samples (5 mg) were mixed with 100 mg of KBr in a pestle and mortar. Pellets were prepared under a hydraulic pressure of 600 kg/cm 2 . These pellets were again crushed and repelleted. This step was repeated 2-3 times to get better reproducibility. Spectra were taken in the wavelength range 400-4000 cm -1 .

XG-ray diffraction (XRD) analysis
The XG-ray diffraction of the of pristine drug, SA/GE placebo micro beads, drug loaded SA/GE micro beads, SA/GE/KA placebo micro beads and drug loaded SA/GE/KA micro beads were performed by a wide angle XG-ray scattering diffractometer (Panalytical XG-ray Diffractometer, model-X'pert Pro) with CuKα radiation (λ= 1.54060) at a scanning rate of 5 °/min to determine the crystallinity.

Field emission scanning electron microscopy (FESEM) analysis
The morphological characterization of the microbeads was studied by using FESEM (Make: JEOL, Singapore. Model: JEOL JSM-7100F) with an accelerating voltage of 20 kV equipped with an EDAX detector.

Determination of encapsulation efficiency
Percentage of encapsulation efficiency was estimated according to the formula and method reported in previous literature [38]. A known mass of beads (10 mg In vitro drug release studies To study the in vitro drug release kinetics of different formulations were performed at 37 °C using a dissolution tester (Lab India, Mumbai, India) containing of eight baskets. Accurate quantity of dried beads (100 mg) was immersed into 900 ml of phosphate buffer solution pH7.4 at 50 rpm to replicate intestinal fluid atmosphere. At a predetermined time intervals 5 ml of sample withdrawn and 5 ml of fresh Phosphate buffer solution (PBS) was added back to basket to keep total volume constant throughout the experiment. Amount of D-PA released was assayed by using a UV-Vis spectrophotometer at 215.2 nm, and the released drug amount was obtained by using concentration versus absorbance calibration curve.

Drug release kinetics
The drug release kinetics was analyzed by fitting the data in to kinetic models, which include zeroth, first order, Higuchi and Korsmeyer-peppas [39][40][41][42]. Based on the goodness of data fit, the highest correlation coefficient was indicative of best-fit model to describe drug release kinetics [43].

Intercalation kinetics
Intercalation kinetics reveals that the time required for intercalation of D-PA with KA by a rapid ion-exchange process between sodium ions of KA and cations of D-PA molecules. From fig. 2 it was clear that 14.9% of D-PA was intercalated in interlayer of KA within 90 min, and remains constant up to 14h. Therefore we should keep 90 min time for interaction between D-PA and KA to avoid partial interaction in the following experiments.

Effect of pH
The effect of pH on the intercalation of D-PA into the interlayer of KA is displayed in fig. 3. The results reveal that suitable pH range for intercalation of D-PA with KA is 6.0-8.5, because at this range intercalation of D-PA almost linear. At pH below 6.0 there was a decrease in intercalation of D-PA in KA layer. At a low pH, hydronium ions increase, therefore a competition was arise between drug molecules and H + ions of KA layer, thus the system is flocculated; hence, adsorption decreases [44].

Fourier transform infrared (FTIR) spectral analysis
FTIR spectra of sodium alginate (a), gelatin (b), placebo SA/GE microbeads (c), pure drug (d) and drug loaded SA/GE microbeads are displayed in fig. 4. FTIR spectra of SA ( fig. 4. a) shows a characteristic peaks at 3415 cm -1 corresponds to O-H stretching frequency, the peaks arises at 1386 cm -1 and 1594 cm -1 assigned to asymmetric and symmetric stretching frequency of carboxylate group C-O. FTIR spectra of GE ( fig. 4. b) a broad band at 3341 cm -1 corresponded to stretching vibration of N-H group, whereas amide (C = O) stretching vibrations are observed at 1616 cm -1 . Further, the absorption band at 1373 cm -1 assigned to stretching vibrations of C-N bond. FTIR spectra of placebo microbeads ( fig. 4. c) a new band formed at 1612 cm -1 is assigned to C=N stretching vibrations which indicates the interaction between-OH group of SA and NH2 group of GE which confirms the formation of microbeads [36]. The FTIR spectra of pristine D-PA ( fig. 4. d) shows a band at 3473, 2607 and 1627 cm -1 were due to-N-H,-S-H and-COOH stretching vibrations. Comparing the FTIR spectra of placebo microbeads and drug loaded microbeads, in placebo microbeads ( fig. 4. c) a band appears at 1612 cm -1 , whereas this band was shifted to lower wavelength i.e. 1596 cm -1 in drug loaded microbeads ( fig. 4. e), which indicates the interaction occurs between polymer and drug molecule. The band at 2358 cm -1 further indicates the presence of S-H stretching frequency, which confirms the drug present in the drug loaded microbeads [38].

: FESEM images of SA/GE placebo micro beads (a,d), drug loaded SA/GE micro beads (b,e) and drug loaded SA/GE/KA micro beads (c,f) and EDS analysis of SA/GE placebo micro beads (g), drug loaded SA/GE micro beads (h) and drug loaded SA/GE/KA micro beads (i)
The

X-ray diffraction (XRD) analysis
Powder XRD patterns of pristine drug (a), SA/GE placebo micro beads (b), drug loaded SA/GE micro beads (c), SA/GE/KA placebo micro beads (d) and drug loaded SA/GE/KA micro beads (e) were displayed in fig. 8. The XRD pattern of D-PA ( fig. 6. a) shows a characteristic peaks at 2θ of 9.10 °, 17.04 °, 17.64 °, and 30.18 °, represents the crystalline nature. Whereas the crystalline peaks are not completely observed in drug loaded SA/GE micro beads ( fig. 6. c), this suggests that the drug is dispersed at molecular level in the polymer matrix. The XRD patterns of SA/GE/KA placebo micro beads ( fig. 6. d) shows a characteristic peaks at 11.98 ° and 20.06 °, indicates KA is present in the microbeads. Whereas these peaks were observed in the drug loaded SA/GE/KA micro beads ( fig. 6. e) but the basal space of these peaks were increased this indicates the KA intercalates with the drug molecules.

FESEM and EDS analysis
In order to examine the surface morphology and chemical composition of KA loaded SA/PEG microbeads, surface SEM image and EDS analysis were performed. Fig. 7 presents the topographical images of placebo SA/GE microbeads ( fig. 7. a and d), drug loaded SA/GE microbeads ( fig.  7. b and e) and drug loaded SA/GE/KA microbeads ( fig. 7. c and f). The SEM image of placebo SA/GE microbeads shows spherical, uniform and smooth surface with detectable pores. Whereas the drug loaded SA/GE microbeads shows pores on the surface this indicates drug should be loaded in the beads. The SEM image of drug loaded SA/GE/KA microbeads shows higher roughness compared with the drug loaded microbeads this is due to the hydrophilic character of clay which facilitates the miscibility of KA with SA/PEG blend matrix.
Higher the KA contents in the polymer matrix higher the surface roughness in the microbeads. This is due to polymer matrix molecules that migrate toward the negative charge of the clay and entered into the clay layers and finally form a porous structure. This confirms the kaolin loaded in the microbeads [45].
The energy-dispersive X-ray spectra (EDS) analysis shows the presence of C, N, O elements in the placebo SA/GE microbeads ( fig. 7. g) and drug loaded SA/GE/KA microbeads ( fig. 7. h). On comparing the EDS spectra of placebo SA/GE microbeads and drug loaded SA/GE/KA microbeads in both spectra nitrogen peaks were observed indicating the presence of gelatin polymer. But the nitrogen peak in drug loaded SA/GE/KA microbeads is more intense than the peak present in placebo SA/GE microbeads and also additional Sulphur peak was observed in drug loaded SA/GE microbeads, which confirms the drug present in drug loaded SA/GE microbeads. Whereas the additional signals corresponding to the Si and Al elements were detected in the drug SA/GE/KA microbeads ( fig. 7. i). This confirms the KA present in the microbeads.

Determination of % encapsulation efficiency (%EE)
The percentages of encapsulation efficiency (%EE) of D-PA loaded microbeads are displayed in table 1. The values are (%EE) lies in between 52.1% to 76.3%. This indicates the %EE dependence on formulation parameters which include percentage of blend composition, concentration of GA and KA. The percentage of EE in formulations of F1, F2 and F3 decreases with the increase of gelatin composition in the blend composition, the decrease of encapsulation is due to gelatin might have assisted the diffusion of drug particles into the external surface to form pores in the matrix. As the concentration of glutaraldehyde increases in the formulations of F5, F6 and F7 the %EE decreases, this is due to the crosslinking density increases the microbeads will become more rigid, thereby the crosslinker reduced the sizes of voids in the matrix. The formulations F8,F9,F10 with the increasing the amount of KA in the polymer matrix, the percentage of D-PA encapsulation efficiency increased this is due to kaolin has large specific area in its structure, good absorption capacity and develops hydrogen bonding between D-PA and hydroxyl groups present on surface of KA.

In vitro drug release studies
Based on the results of effect of pH on intercalation of clay and drug, the cumulative percentage of drug release was studied for all the formulations in pH 7.4 buffer solution at 37 °C. In vitro drug release studies were discussed in terms of effect of polymer, effect of kaolin clay, drug variation and crosslinker variation.

Effect of polymer variation
The effect of polymer blend content was studied with constant loading of D-PA and glutaraldehyde. The drug release profiles of F2, F4 and F5 are 80.4%, 87% and 92% respectively are displayed in fig. 8. This suggests that drug release rate increases with increasing content of sodium alginate, this is due to hydrophilic nature of sodium alginate [46], whereas the gelatin concentration increases the drug release rate decreases, this is due to the rigid nature of SA/GE blend component and the possible hydrogen-bonding interactions between −COO− of sodium alginate and N-H group of gelatin.

Effect of crosslinking agent
To investigate the effect of crosslinker on in vitro release profiles was studied by plotting the cumulative % of drug release data against time for varying concentration of GA (i.e., 2.0, 4.0 and 6.0 ml) with constant of D-PA and polymer blend matrix are displayed in fig.   9. The drug release rate was higher when the microbeads containing small concentration of GA (i.e., 2 ml), whereas the release rate was lower when GA concentration was increased (i.e., 6 ml) in the polymer matrix. This is due to increased stiffness in polymer chains and reduced porosity of microbeads at higher crosslink densities, thereby hindering the transport of D-PA through the microbeads. The release profile shows a burst release with in 2 h and extended the release over the remaining time is due to swelling of the hydrophilic matrix. From the cumulative release data, it was found that the formulation which contains more amount of drug (F3) showed higher release rates and the formulation which contains lower amount of drug (F1) showed lower release rate. This indicates that release rates vary depending on the amount of drug in the matrices, i.e., the release rate was found to be significantly faster at higher amounts of drug this is due to osmotic pressure is high in higher concentration of drug which leads to quick diffusion of drug from the matrix to dissolution media [47]. The release rate was slower in F1, this might be due to the availability of extra free void spaces through which fewer drug molecules will transport [48].

Effect of kaolin clay
To understand the effect of KA on in vitro release profiles was studied by plot the cumulative % of drug release data against time for varying amounts of KA (i.e., 400, 600 and 800 mg) with constant D-PA and polymer blend matrix are displayed in fig. 11. The cumulative release rate of D-PA from microbeads containing KA is decreases as the concentration of KA increases, this is due to intercalated drug cannot be exchanged completely in ion exchange process with phosphate ions of the buffer solution and also the electrostatic attraction between amino groups of D-PA and anionic groups of KA layer which leads to incomplete release process.

Drug release kinetics
To find out the drug release mechanism, the data obtained from in vitro drug release studies in PBS (7.4) were fitted into different kinetic models including zero order, first order, Higuchi and Korsmeyer-Peppas models which are empirical in nature, the results of rate constant and correlation coefficient (r 2 ) of all formulations are displayed in Where, Mt/Mα represents the fractional drug release at time t, k is a constant characteristic of the drug-polymer system and n is the release exponent indicating the type of drug release mechanism. The values n are obtained in the range of 0.557-0.693 indicating that the drug release mechanism followed Non-Fickian type of diffusion process. These results, along with correlation coefficients 'r 2 'are presented in Table.2. A similar observation was reported by E. Yilmaz et al., [49] from the Preparation and characterization of pHsensitive semi-interpenetrating network hybrid hydrogels with sodium humate and kaolin.

CONCLUSION
A simple ionotropic gelation method was used to fabricate D-PA loaded SA/GE/KA microbeads. Microbeads formation was confirmed by FTIR spectroscopy. X-RD reveals that the KA should be intercalated with the drug and also it confirms the molecular level dispersion of D-PA into microbeads. Scanning Electron Microscopy (SEM) studies reveal that the beads were in spherical shape with some wrinkled depressions on the surface. The in vitro release study indicates the D-PA released in a controlled manner. The in vitro release kinetics was assessed by different empirical equations and the data supported a Non-Fickian diffusion mechanism. Based on the results, Kaolin intercalated microbeads are promising material for drug carrier in emerging drug delivery systems.