International Journal of Biological Macromolecules 69 (2014) 514–522
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
In vitro and in vivo evaluation of novel interpenetrated polymer network microparticles containing repaglinide Raghavendra V. Kulkarni a,∗ , Foram S. Patel a , H.M. Nanjappaiah b , Akram A. Naikawadi c a b c
Department of Pharmaceutical Technology, BLDEA’s College of Pharmacy, BLDE University Campus, Bijapur 586 103, India Department of Pharmacology, BLDEA’s College of Pharmacy, BLDE University Campus, Bijapur 586 103, India Department of Pharmacology, Sri. B. M. Patil Medical College and Research Centre, BLDE University, Bijapur 586 103, India
a r t i c l e
i n f o
Article history: Received 16 March 2014 Received in revised form 15 May 2014 Accepted 5 June 2014 Available online 17 June 2014 Keywords: Interpenetrated polymer network Microparticles Repaglinide Drug release
a b s t r a c t Interpenetrated polymer network (IPN) microparticles of sterculia gum and sodium alginate loaded with repaglinide were developed by ionic gelation and emulsion crosslinking method. The drug entrapment efficiency was as high as 91%. FTIR and TG analyses confirmed the crosslinking and IPN formation. Microparticles have demonstrated the drug release up to 24 h depending upon type of crosslinking agents; the glutaraldehyde treatment of ionically crosslinked microparticles has resulted in decreased drug release rate. The in-vivo anti-diabetic activity performed on streptozotocin induced diabetic rats indicated that the pristine repaglinide has shown maximum percentage reduction of elevated blood glucose within 3 h and then the percentage reduction in blood glucose was decreased. In the case of rats treated with KA8 IPN microparticles, percentage reduction of elevated glucose was slow as compared to pristine drug within 3 h, but it was gradually increased to 81.27% up to 24 h. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the development of controlled drug delivery systems has realized the progress in terms of clinical efficacy and patient compliance. Of the several controlled drug delivery systems, microparticles are gaining importance. On the oral administration, the microparticles spread uniformly over the gastrointestinal tract and release the drugs more uniformly. This avoids the local high drug concentration and toxicity [1,2]. Polysaccharides are the choice of excipients for developing the controlled release microparticles; since they are non-toxic, biodegradable and easily available [3,4]. Though the polysaccharides have limitations in their reactivity and processibility, they are still being used in drug delivery applications following modification by cross-linking, blending etc. Many efforts have been reported to overcome these limitations by functional modification of polysaccharides. Among them, developing interpenetrating polymer network (IPN) is of the research interest for polymer and pharmaceutical scientists [5,6]. IPN is normally a blend of two polymers in the network form; at least, one of the polymers is synthesized and/or crosslinked in the presence of other polymer [7,8]. To date several natural and synthetic polymers have been utilized for preparing IPNs [9] for many
∗ Corresponding author. Tel.: +91 9845619296. E-mail address: pharma [email protected] (R.V. Kulkarni). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.011 0141-8130/© 2014 Elsevier B.V. All rights reserved.
applications including artificial implants, dialysis membranes and drug delivery devices [10]. IPNs are competent of delivering drugs for an extended period; in such drug delivery systems, extent of crosslinking can be monitored to the control of drug release [11,12]. Sodium alginate (SA) is a natural polysaccharide obtained from marine brown algae (MW ≈ 240,000). It is a hydrophilic salt of alginic acid consisting of two uronic acids, -d-mannuronic acid (M) and ␣-l-glucouronic acid (G). It is composed of homopolymeric blocks MM or GG [13]. SA is widely used as gelling agent in the food industries. It forms a gel in the presence of multivalent cations. This gel formation is due to ionic crosslinking of alginate by the replacement of sodium ions with multivalent cations. Thus crosslinked alginate has wide applications in the controlled release of drugs [14,15]. Sterculia gum (SG), also called as karaya gum is a water-soluble high molecular weight (MW ≈ 9,500,000) colloid prepared from the exudates of Sterculia urens tree. It contains three different types of chains; one chain (constituting 50% of the total polysaccharide) contains repeating units of four galacturonic acid residues containing -d-galactose branches and l-rhamnose residues at the reducing end of the unit. A second chain (17% of the polysaccharide) contains an oligorhamnan having d-galacturonic acid branch residues and interrupted occasionally by a d-galactose residues. A third chain (33% of the polysaccharide) contains d-glucuronic acid residues. It constitutes about 13–26% galactose, 15–30% rhamnose and approximately 40% uronic acid residues [16,17]. It is used as a
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bulk laxative [18]. The SG has been reported as a controlled release matrix and has shown better mucoadheshion as compared to guar gum [19]. Repaglinide (RPG) is an oral anti-hyperglycemic drug used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) that acts by stimulating -cells of the pancreas to release insulin. Though RPG shows excellent anti-diabetic activity, it has short half-life (1 h) and low bioavailability (50%). It is rapidly and completely absorbed in the gastrointestinal tract (GIT); however, the absorption is poor from the upper part of the GIT. In addition, it produces hypoglycemia after the oral administration [20]. In view of the fact that this drug is used for long-term treatment, patient compliance is a major concern [21]. The effective control of type II diabetes requires administration of RPG at a dose of 0.5–4 mg three times daily [22]. The drugs, which absorb without difficulty from GIT and have short half-life, are expected to eliminate rapidly from the body and require repeated administration, which may lead to the increased drug toxicity and patient non-compliance. To circumvent this problem, the controlled release systems are being designed to maintain constant drug concentrations in the plasma for longer period [23]. Therefore, the objective of the study was to develop and evaluate dual crosslinked interpenetrating network hydrogel microparticles using SG and SA for controlled release of RPG. The prepared IPN microparticles were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction studies, scanning electron microscopy and evaluated for in vitro drug release behavior and in vivo anti-diabetic activity in wistar rats. 2. Materials and methods
515
was emulsified into light liquid paraffin to form water-in-oil (W/O) emulsion using a high speed homogenizer (T 25 Digital Ultra turrax homogenizer, IKA, Germany) in a beaker containing light liquid paraffin and span 80. The solution of CaCl2 or BaCl2 or AlCl3 was added slowly and stirred for 4 h at 500 rpm. The microparticles were filtered, washed with n-hexane and distilled water to remove the oil as well as excess amount of surfactant and they were dried in a hot air oven at 40 ◦ C for 12 h. Further, these ionically crosslinked microparticles were covalently crosslinked by transferring to a methanolic solution containing glutaraldehyde (GA) and 1 N HCl and allowed to react for 30 min at 50 ◦ C. Thus, the dual crosslinked IPN microparticles were washed, dried and stored in a desiccator for further analysis. Formulation composition is shown in Table 1. 2.3. Scanning electron microscopic (SEM) analysis The SEM analysis was performed using required magnification at room temperature. The acceleration voltage used was 10 to 20 kV with the secondary electron image as a detector. The IPN microparticles were coated with platinum to a thickness of about 300 A˚ under an inert atmosphere using a sputter coater module (Edward S 150, UK) in a vacuum evaporator. The coated microparticles were examined under scanning electron microscope (JSM-6360, Jeol, Japan). 2.4. Particle size analysis An optical microscopic method was used to evaluate the size of IPN microparticles. An eyepiece micrometer was used for this purpose, which was calibrated using stage micrometer. Size of 100 particles was measured for each batch and average particle size was calculated.
2.1. Materials 2.5. Drug encapsulation efficiency (DEE) Repaglinide (RPG) was a gifted sample from Torrent Pharmaceuticals (Ahmadabad, India). Sterculia gum (SG), sodium alginate (SA), liquid paraffin (light), span 80, calcium chloride (CaCl2 ), barium chloride (BaCl2 ) and aluminum chloride (AlCl3 ) and glutaraldehyde (GA; 25%) were purchased from HiMedia Laboratories Pvt. Ltd., (Mumbai, India), streptozotocin (STZ) was purchased from CDH Pvt. Ltd. (New Delhi, India). Double distilled water was used throughout the study. All other chemicals were used without further purification. 2.2. Preparation of IPN microparticles
An accurately weighed amount of IPN microparticles were allowed to swell in phosphate buffer of pH 7.4 (100 ml) for 24 h. The swollen microparticles were crushed and the solution was gently heated at 40 ◦ C for 1 h to extract the drug and then centrifuged to remove the polymeric wreckage. The clear supernatant liquid was taken and subjected for drug content estimation using UV–visible spectrophotometer (Model UV-1800, Shimadzu, Japan) at 243 nm. The encapsulation efficiency was calculated using the following equation: DEF =
The IPN microparticles of SG and SA were prepared by emulsioncrosslinking method. The SG and SA polymers were dissolved in double distilled water by stirring to obtain a homogeneous solution. The required amount of RPG was dispersed in polymeric solution and stirred well for 30 min. The drug loaded polymeric solution
Experimental drug content × 100 Theoretical drug content
(1)
2.6. Fourier transform infrared (FTIR) analysis FTIR analysis was performed to study crosslinking reaction and stability of drug within the IPN matrix. The spectra of drug and drug
Table 1 Composition of IPN microparticles. Ingredients
KA1
KA2
KA3
KA4
KA5
KA6
KA7
KA8
Drug (mg) Sterculia gum (% w/v) Sodium alginate (% w/v) Distilled water (ml) Liquid paraffin light (ml) Span-80 (% w/v) CaCl2 (% w/v) BaCl2 (% w/v) AlCl3 (% w/v) Glutaraldehyde (ml) 1 N HCL (ml)
50 1 3 10 100 0.5 8 – – – –
50 2 2 10 100 0.5 8 – – – –
50 3 1 10 100 0.5 8 – – – –
50 2 2 10 100 0.5 – 8 – – –
50 2 2 10 100 0.5 – – 8 – –
50 2 2 10 100 0.5 8 – – 1 1
50 2 2 10 100 0.5 – 8 – 1 1
50 2 2 10 100 0.5 – – 8 1 1
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loaded IPN microparticles were taken on FTIR instrument (FTIR8400S, Shimadzu, Japan) in the range of 4000–500 cm−1 . 2.7. Thermogravimetric analysis (TGA) TG analysis was performed using a microcalorimeter (Diamond TG/DTA, Perkin Elmer, USA) under a dynamic argon atmosphere flowing at a rate of 50 ml/min and at a heating rate of 10 ◦ C/min. in the temperature range 0–400 ◦ C. 2.8. Differential scanning calorimetric (DSC) analysis The DSC analysis of pristine drug, drug loaded IPN microparticles and drug free IPN microparticles was carried out. The thermal analysis was performed by recording thermograms for 5–15 mg samples at a heating rate 10 ◦ C/min in a temperature range 0 ◦ C to 300 ◦ C under argon flow rate of 25 ml/min. using a microcalorimeter (DSC Q20 V24.4 Build 116, TA Instruments, USA). 2.9. X-ray diffraction (XRD) studies The XRD analysis of pristine drug, drug free IPN microparticles and drug loaded IPN microparticles was performed in order to know the crystallinity of drug after formulation. The study was carried out using a benchtop X-ray diffractometer (Rigaku Miniflex II, Japan) in the 2 range 0–80◦ . 2.10. In-vitro drug release behavior The in-vitro release rate of RPG form IPN microparticles was carried out using dissolution apparatus (USP-XXIII Electrolab TDT 08L, Mumbai) consisting 900 ml of buffer of pH 1.2 for the first 2 h and phosphate buffer of pH 7.4 till end of the study at 37 ± 0.5 ◦ C and 100 rpm. Drug release study was carried out for 24 h. The 5 ml aliquots were withdrawn at different time intervals and replaced with an equal amount of fresh dissolution medium. Further, the samples were filtered through a 0.45 mm membrane filter and the amount of drug released was determined using UV–visible spectrophotometer (Model UV-1800, Shimadzu, Japan) following suitable dilutions at 243 nm. 2.11. In-vivo anti-diabetic activity Albino rats of wistar strain (150–250 g) were selected and kept in standard polypropylene cages under controlled room temperature. The rats were fed with standard laboratory diet and water. Animals were kept under fasting for 24 h with water ad libitum during the experiments. Experimental protocol was approved by the institutional animal ethics committee (IAEC), registered under CPCSEA. The rats were made diabetic by intraperitoneal injection of freshly prepared streptozotocin solution at a dose of 60 mg/kg dissolved in water for injection. After 48 h of administration, the rats with fasting blood glucose of 180 mg/dl or more were taken as diabetic and used for further studies. The rats were divided into four groups of six rats each and treated as follows, Group I: normal control rats, Group II: diabetic control rats, Group III: standard rats (treated with pristine repaglanide), Group IV: test rats (treated with KA8 IPN microparticles). The pristine drug and KA8 IPN microparticles were administered orally as suspension in 2% acacia. Blood samples were collected from retro-orbital plexus of each rat under mild anesthesia at 0, 1, 3, 6, 9, 12 and 24th hour after administration and they were analyzed for blood glucose.
Table 2 Average size, drug entrapment efficiency, diffusion coefficients (D) and release parameter (n) of the IPN microparticles. Microparticles
Average size (m)
KA1 KA2 KA3 KA4 KA5 KA6 KA7 KA8
52.12 58.35 61.52 54.56 51.85 55.41 53.25 49.75
± ± ± ± ± ± ± ±
8.13 7.38 3.78 4.25 9.45 9.32 6.42 7.21
DEE (%) 87.83 83.64 81.10 84.59 89.36 85.87 86.71 91.70
± ± ± ± ± ± ± ±
3.88 8.66 7.36 2.16 4.38 4.14 4.32 6.75
D (cm2 /s)
n
6.01 × 10−6 6.21 × 10−6 6.36 × 10−6 5.87 × 10−6 5.23 × 10−6 5.01 × 10−6 4.86 × 10−6 4.23 × 10−6
0.68 0.64 0.61 0.66 0.68 0.73 0.77 0.79
All the values are average of three determinations. ± Indicates SD values. D indicates diffusion coefficient values and n values indicate release mechanism.
2.12. Histopathology of rat pancreas At the end of the study, rats were sacrificed, pancreas were isolated, washed with normal saline and quickly fixed in 10% formalin. The pancreatic tissue was processed according to standard histopathological technique. The tissue was dehydrated in ascending degrees of isopropyl alcohol, cleaned in xylene and impregnated in paraffin wax. The 5 m thick paraffin wax boxes were made, sections were used for cutting microtome and stained with hematoxylin–eosin method and then observed under binocular light microscope (CXRIII, Labomed, India). All the results were expressed as ±SD. Statistical evaluation was done using one-way analysis of variance (ANOVA), followed by Student’s t-test. Differences in mean values were considered significant at p < 0.05. 3. Results and discussion 3.1. Preparation of IPN microparticles The IPN microparticles of SG and SA were prepared by emulsion crosslinking method for the controlled release of RPG. On addition of CaCl2 or BaCl2 or AlCl3 solutions to the polymeric solution of SG and SA, ionic cross-linking occurs between two SA chains and different parts of the same polymer chain. The exchange of Na+ ions occurs with Ca2+ or Ba2+ or Al3+ ions at the carboxylate site and a second strand of SA can also be connected to the same by forming a link in which the counter ions are attached to two or three SA strands jointly. Further, on treating these ionically crosslinked microparticles with GA, an acetal structure has been formed between the CHO groups of GA and OH groups of SA and SG chains to form an IPN including SA and SG, thus making the polymer matrix insoluble (Fig. 1). 3.2. SEM and particle size analysis The shape and surface morphology was examined by SEM analysis; IPN microparticles possess smooth surface as shown in Fig. 2. The average size of IPN microparticles was in the range of 49.75 to 61.52 m (Table 2). Size of the microparticles was increased as the concentration of SA was decreased; the size was also depended upon the type of ionic crosslinking agents. AlCl3 produced smaller microparticles than BaCl2 , which in turn produced smaller microparticles than CaCl2 . During cross-linking, the polymer network might have undergone syneresis resulting in formation of smaller microparticles at higher cross-link densities. Similar results have been reported earlier [24]. As compared to ionically cross-linked microparticles (KA3–KA5), the size of the dual crosslinked microparticles (KA6–KA8) was smaller; it may be due to rapid shrinking of the polymer matrix due to the formation of covalent cross-links
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Fig. 1. Schematic representation of the prepared IPNs.
between the polymer chains resulting in rigid IPN network. This is in agreement with the previously published results [25]. 3.3. Drug encapsulation efficiency DEE of the IPN microparticles was in the range of 81.10% to 91.70% (Table 2). The DEE was decreased with decrease in SA concentration. DEE of the microparticles prepared with Al3+ ions was highest than those prepared with Ba2+ , which in turn was higher than the microparticles prepared with Ca2+ ions. In case of microparticles prepared with Ca2+ ions, the polymer network might be loose with larger pores that results in leakage of entrapped drug into the external medium from polymer matrix during the preparation, leading to lower DEE. Whereas, in case of microparticles prepared with Al3+ ions, the polymer matrix is rigid and leakage
of drug from polymer matrix is low leading to higher DEE. The DEE of microparticles prepared by ionic cross-linking was lower than those prepared by dual cross-linking (both ionic and covalent crosslinking), it may be due to the formation of more stiffer matrix, that reduces the leakage of entrapped drug from the IPN matrix. Similar observations were also reported earlier [26]. 3.4. FTIR analysis The FTIR spectroscopy was used to confirm the crosslinking and IPN formation. Fig. 3 displays the FTIR spectra of SA (A), SG (B) and placebo IPN microparticles KA8 (C), pristine RPG (D) and drug loaded KA8 microparticles (E). In case of SA (A), the broad peak appearing at ∼3400 cm−1 is due to stretching vibrations of hydroxyl groups. The peaks at 1618 and 1683 cm−1 are assigned to
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Fig. 2. Scanning electron microscopic photographs of IPN microparticles (A) and surface morphology (B).
the deformation of carbonyl groups of SA; the peak appearing at 2924 cm−1 is due to the C H stretching of cyclic aldehyde and the peaks appeared at 1031 and 1099 cm−1 are due to C O stretching of alcoholic groups. In the spectra of SG (B), the broad peak appearing at 3425 cm−1 is due to stretching vibrations of hydroxyl groups. The peak appearing at 1630 cm−1 corresponds to deformation of carbonyl group of SG; the peak appearing at 2924 cm−1 is due to the C H stretching of cyclic aldehyde and the peaks appeared at 1043 and 1149 cm−1 are due to the C O stretching of alcoholic groups. While in the spectra of placebo IPN microparticles KA8 (C), the peak at ∼3300 cm−1 is due to stretching vibrations of OH groups of the polysaccharides; sharp peaks appearing at 1687 and 1627 cm−1 correspond to carbonyl functional groups of polysaccharides. The peak at 1438 cm−1 is due to symmetric stretching of the carboxylate groups, whereas the peak appearing at 1036 cm−1 represents the C O C stretching vibrations. The relatively sharp peak appearing at 1215 cm−1 is corresponding to the formation of acetal structures due to reaction between hydroxyl groups of SG & SA and aldehyde groups of GA. During crosslinking, GA reacts with hydroxyl groups of SG and SA in the presence of each other to form interpenetrated polymer network through acetal structures. This could be further supported by the presence of sharp and relatively high intensity peak at 2933 cm−1 due to CH2 groups of the alkyl chain formed by crosslinking [27]. This confirms the crosslinking and IPN formation. FTIR spectroscopy was also used to know the drug stability within IPN matrix. The pristine RPG (D) exhibits peak at 3308 cm−1 is due to stretching of hydroxyl groups. The peak at 2928 cm−1 is due to aliphatic C H stretching, peak at 1687 cm−1 is due to C O stretching of ketone and the peak at 1637 cm−1 is due to carbonyl
Fig. 3. FTIR spectra of SA (A), SG (B) and placebo IPN microparticles KA8 (C), repaglinide (D) and drug loaded KA8 microparticles (E).
groups. Whereas in the spectra of drug loaded KA8 microparticles (E), same characteristic peaks related to pristine RPG were observed with slight deviations. This indicates the stability of drug within IPN matrix. 3.5. TG analysis The typical thermograms of samples are shown in Fig. 4. In case of ionically crosslinked microparticles (KA2, KA4 and KA5), the decomposition of matrix started at lower temperature and we observed a sudden mass loss. KA2 microparticles started decomposing after 100 ◦ C and 24.71% mass loss up to 100 ◦ C is due to loss of loose and bound water in the matrix. A sharp mass loss of 49.08% was observed between 100 ◦ C and 200 ◦ C and it reached a value of 75.32% at 400 ◦ C. This may be attributed to the decomposition of polymer matrix. The decomposition of KA4 and KA5 microparticles started after 100 ◦ C and mass loss (21.67% and 12.38%) up to 100 ◦ C is due to the loss of water present in the matrix. A 45.66% mass loss was observed between 100 ◦ C and 200 ◦ C and it reached a value of 68.42% and 61.19%, respectively, at 400 ◦ C. Thus, the thermal stability of microparticles prepared with AlCl3 was more as compared to those prepared with BaCl2 , which in turn was more thermally stable than the microparticles prepared with CaCl2 . Whereas, in case of dual crosslinked IPN microparticles (KA6, KA7 and KA8), the matrix decomposition started at higher temperature, mass loss was found to be constant and percent residual mass was higher than the ionically crosslinked microparticles. The
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Fig. 6. X-ray diffractograms of pristine repaglinide (A), drug free KA8 microparticles (B), and drug loaded KA8 microparticles (C). Fig. 4. TGA thermograms of KA2 (A), KA4 (B), KA5 (C), KA6 (D), KA7 (E) and KA8 (F) IPN microparticles.
KA6, KA7 and KA8 microparticles have shown mass loss of 59.28%, 51.17% and 40.30%, respectively, at 400 ◦ C. The dual crosslinked microparticles have shown greater thermal stability than the ionically crosslinked microparticles. In case of dual crosslinked IPN microparticles, as the polymeric chains are strongly entangled together, the thermal stability is higher. This suggests the formation of IPN comprising of SG, SA and crosslinking agents. 3.6. DSC analysis The DSC analysis of pristine RPG, drug-free KA8 microparticles and drug-loaded KA8 microparticles was carried out and the results are depicted in Fig. 5. The drug-free IPN microparticles have shown an endothermic peak at 121 ◦ C, whereas, drug-loaded IPN microparticles showed an endothermic peak at 117 ◦ C. Thus, appearance of endothermic peak at lower temperature in case of drug-loaded IPN microparticles may be due to the formation of loose matrix because of creation of extra free space after drug loading. The pristine RPG has shown a sharp endothermic peak at 136 ◦ C due to melting of the drug, but this endothermic peak is not observed in drug-loaded IPN microparticles. This suggests the uniform and amorphous dispersion of drug in the IPN matrix.
Fig. 5. DSC thermograms of pristine repaglinide (A), drug free KA8 microparticles (B) and drug loaded KA8 microparticles (C).
3.7. XRD studies The X-ray diffractograms of pristine RPG (A), drug free KA8 microparticles (B) and drug-loaded KA8 microparticles (C) are presented in Fig. 6. Pristine RPG has shown distinctive intense peaks between the 2 of 12◦ and 25◦ due to crystallinity of drug. Whilst, in case of drug free and drug loaded IPN microparticles, no intense peaks related to drug were noticed between the 2 of 12◦ and 25◦ . But in the diffractogram of drug-loaded KA8 microparticles, few peaks were appeared between the 2 of 25◦ and 30◦ that may be due to surface adhered drug. The diffractograms of both drug-free and drug loaded IPN microparticles are almost one and the same; this indicates the amorphous dispersion of drug in IPN matrix. 3.8. In-vitro drug release studies The in-vitro drug release behavior of IPN microparticles was performed using dissolution rate test apparatus in 0.1 N HCl (pH 1.2) for 2 h and in phosphate buffer (pH 7.4) until end of the study. The drug release profiles of RPG are shown in Fig. 7. Results indicated that the drug release was continued up to 24 h depending upon the formulation variables. The drug release depended upon the types of crosslinking agents; the release was slow from the microparticles, which were prepared with Al3+ as compared to those prepared with Ba2+ ions, which in turn was slower than the microparticles prepared with Ca2+ ions. The GA treatment of these microparticles
Fig. 7. In vitro release profiles of repaglinide from IPN microparticles.
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has further resulted in decreased drug release. As the concentration of SA was decreased, drug release was increased. The ionically crosslinked microparticles discharged the drug quickly while, dual crosslinked IPN microparticles extended the drug release for longer period. This may be due to decrease in free volume of the IPN matrix at higher crosslinking, which obstructs the movement of drugs through the IPN matrix [25]. The diffusion coefficients of drug from IPN microparticles were computed using the following equation [28]:
D=
r 6M∞
2
(2)
while is the slope of linear portion of the plot of Mt /M∞ versus t1/2 , r is radius of microparticles and M∞ is the total quantity of drug loaded. The diffusion coefficients were calculated based on the Fickian diffusion model and the D values are listed in Table 2. The diffusion coefficient of drug was increased with decrease in the concentration of SA. The D values were depended upon the type of crosslinking agent. For example, the D value of KA2 microparticles prepared with Ca2+ ions was higher than KA4 microparticles, which were prepared with Ba2 + ions, and the latter was higher than the KA5 microparticles, which were prepared with Al3+ ions. The Ba2+ ions can produce stronger PIN matrix than Ca2+ ions, whilst Al3+ being a trivalent ion produces still stronger matrix with higher crosslink density than the Ca2+ and Ba2+ ions. Similar results were also reported earlier [24]. Further, D values were lower for the microparticles prepared with dual crosslinking as compared to ionically crosslinked microparticles. It is evident from the study that the increased crosslinking resulted in increased rigidity of IPN network, thus decreasing the drug diffusion. To know the mechanism of drug release, dissolution data was fitted into the following empirical equation [29]: Mt = Kt n M∞
Fig. 8. Blood glucose level of rats treated with pristine repaglinide and repaglinide loaded KA8 IPN microparticles.
(3)
where Mt is quantity of drug released at time t, and M∞ is the total quantity of drug loaded into microparticles, n values indicate the type of release mechanism. The n values are computed for the IPN microparticles and listed in Table 2. The value of n depends upon the type of crosslinking agent; it increases with increased crosslink density. The computed n values indicated that the mechanism of drug release followed non-Fickian transport. Further, KA8 IPN microparticles were selected for in vivo anti-diabetic activity because this formulation has demonstrated satisfactory in vitro drug release behavior. 3.9. In-vivo anti-diabetic activity The pristine RPG and RPG loaded IPN microparticles (KA8) were administered to the streptozotocin induced diabetic rats at a dose of 1.44 mg/kg bw after converting human dose to rat dose. Blood glucose level in diabetic rats were raised nearly 4–5 folds as compared to normal control group rats. The increase in glucose level in diabetic control group was found to be significant (p < 0.05) as compared to normal control group. The rats of normal control group (Group I) have shown blood glucose level in the range of 78.83 to 82.33 mg/dl. Rats of diabetic control group (Group II) have shown blood glucose level in the range of 352.2 to 373.33 mg/dl. Rats of Group III, who received pristine RPG have shown blood glucose level in the range of 127 to 280.8 mg/dl, whereas rats of Group IV, who received RPG loaded KA8 IPN microparticles have shown blood glucose level in the range of 61 to 325.8 mg/dl up to 24 h as shown in Fig. 8. In case of rats treated with pristine RPG, a sudden reduction in blood glucose level was observed up to 3 h; afterwards the blood
Fig. 9. Percentage reduction of blood glucose in rats treated with pristine repaglinide and repaglinide loaded KA8 IPN microparticles.
glucose level was recovered. The percentage reduction of blood glucose was 54.9% at the end of 3 h and gradually it decreased to 18.99% at the end of 24 h. It indicates that the pristine drug has rapidly absorbed, showed action and declined in the plasma; it may be attributed to the shorter biological half-life of RPG. While in case of rats treated with KA8 IPN microparticles, the percentage reduction in blood glucose was slow as compared to pristine RPG up to 3 h; however, percentage glucose reduction was attained above 25% within 1 h and maintained up to 24 h. A 25% reduction in blood glucose is considered to be significant hypoglycemic affect as reported earlier [30]. In our study, the percentage glucose reduction was 34.27% at the end of 3 h, and it was gradually increased to 81.27% at the end of 24 h (Fig. 9). This could be due to the slow release and absorption of RPG over longer period of time [31]. This suggests the controlled release of RPG from IPN microparticles over a longer period as compared to pristine RPG. 3.10. Histopathology of rat pancreas Histopathological evaluation of rat pancreas is shown in Fig. 10 and Table 3. The normal control rats show normal cells in the islets of Langerhans and did not show any pathological anomaly. The diabetic control rats showed higher extent of pathological changes like necrosis of islets, hemorrhages and cellular
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Fig. 10. Histopathology of pancreas of normal control rat (A), diabetic control rat (B), rats treated with pristine repaglinide (C) and rats treated with KA8 IPN microparticles (D).
Table 3 Histopathology of rat pancreas. Sr. no.
Parameters
Normal control rats
Diabetic control rats
Repaglinide treated rats
Rats treated with IPN microparticles
1 2
Necrosis Nucleus changes/disappearance of nucleus Karyolysis Size of islets Dilation of large vessels Congestion of large vessels Any other remarks
0 0
2 2
2 2
0 0
0 Normal 0 0 0
1 Reduced 2 2 Islet cell hyperplasia
1 Normal 1 1 Congested vessels
0 Normal 1 0 0
3 4 5 6 7
Scores: 0: nil, 1: mild, 2: moderate, 3: severe, 4: very severe.
infiltration, and reduced dimensions of islets. The rats treated with pristine RPG showed partial reinstatement of normal cellular population of islets. The damage of pancreas in diabetic control rats and partial restoration of ˇ cells was more effectively observed in rats treated with KA8 IPN microparticles. It was found that the KA8 IPN microparticles were more effective than the pristine RPG.
reduction in blood glucose within 3 h and afterwards the percentage reduction in blood glucose was decreased. While in case of rats treated with KA8 IPN microparticles, the percentage reduction in glucose was initially slow as compared to pristine RPG, but it was gradually increased to 81.27% at the end of 24 h. It can be concluded from the study that, the developed IPN microparticles are versatile delivery systems for RPG, which could release the drug in a controlled manner for the effective treatment of diabetes mellitus.
4. Conclusions IPN microparticles of SG and SA were successfully developed by emulsion crosslinking method for the controlled release of RPG. The prepared IPN microparticles were spherical with entrapment efficiency as high as 91%. The ionically crosslinked microparticles discharged the drug quickly, while dual crosslinked microparticles extended the release of drug for 24 h. The anti-diabetic activity indicated that the pristine RPG has shown maximum percentage
Acknowledgements One of the authors (Prof. R V Kulkarni) is grateful to Vision Group on Science & Technology (VGST), Department of Science & Technology, Government of Karnataka, India for financial assistance through major grant K-FIST (Level-II) programme 2012-13. Authors are also thankful to Dr. B.G. Mulimani, Vice Chancellor,
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
In vitro and in vivo evaluation of novel interpenetrated polymer network microparticles containing repaglinide Raghavendra V. Kulkarni a,∗ , Foram S. Patel a , H.M. Nanjappaiah b , Akram A. Naikawadi c a b c
Department of Pharmaceutical Technology, BLDEA’s College of Pharmacy, BLDE University Campus, Bijapur 586 103, India Department of Pharmacology, BLDEA’s College of Pharmacy, BLDE University Campus, Bijapur 586 103, India Department of Pharmacology, Sri. B. M. Patil Medical College and Research Centre, BLDE University, Bijapur 586 103, India
a r t i c l e
i n f o
Article history: Received 16 March 2014 Received in revised form 15 May 2014 Accepted 5 June 2014 Available online 17 June 2014 Keywords: Interpenetrated polymer network Microparticles Repaglinide Drug release
a b s t r a c t Interpenetrated polymer network (IPN) microparticles of sterculia gum and sodium alginate loaded with repaglinide were developed by ionic gelation and emulsion crosslinking method. The drug entrapment efficiency was as high as 91%. FTIR and TG analyses confirmed the crosslinking and IPN formation. Microparticles have demonstrated the drug release up to 24 h depending upon type of crosslinking agents; the glutaraldehyde treatment of ionically crosslinked microparticles has resulted in decreased drug release rate. The in-vivo anti-diabetic activity performed on streptozotocin induced diabetic rats indicated that the pristine repaglinide has shown maximum percentage reduction of elevated blood glucose within 3 h and then the percentage reduction in blood glucose was decreased. In the case of rats treated with KA8 IPN microparticles, percentage reduction of elevated glucose was slow as compared to pristine drug within 3 h, but it was gradually increased to 81.27% up to 24 h. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the development of controlled drug delivery systems has realized the progress in terms of clinical efficacy and patient compliance. Of the several controlled drug delivery systems, microparticles are gaining importance. On the oral administration, the microparticles spread uniformly over the gastrointestinal tract and release the drugs more uniformly. This avoids the local high drug concentration and toxicity [1,2]. Polysaccharides are the choice of excipients for developing the controlled release microparticles; since they are non-toxic, biodegradable and easily available [3,4]. Though the polysaccharides have limitations in their reactivity and processibility, they are still being used in drug delivery applications following modification by cross-linking, blending etc. Many efforts have been reported to overcome these limitations by functional modification of polysaccharides. Among them, developing interpenetrating polymer network (IPN) is of the research interest for polymer and pharmaceutical scientists [5,6]. IPN is normally a blend of two polymers in the network form; at least, one of the polymers is synthesized and/or crosslinked in the presence of other polymer [7,8]. To date several natural and synthetic polymers have been utilized for preparing IPNs [9] for many
∗ Corresponding author. Tel.: +91 9845619296. E-mail address: pharma [email protected] (R.V. Kulkarni). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.011 0141-8130/© 2014 Elsevier B.V. All rights reserved.
applications including artificial implants, dialysis membranes and drug delivery devices [10]. IPNs are competent of delivering drugs for an extended period; in such drug delivery systems, extent of crosslinking can be monitored to the control of drug release [11,12]. Sodium alginate (SA) is a natural polysaccharide obtained from marine brown algae (MW ≈ 240,000). It is a hydrophilic salt of alginic acid consisting of two uronic acids, -d-mannuronic acid (M) and ␣-l-glucouronic acid (G). It is composed of homopolymeric blocks MM or GG [13]. SA is widely used as gelling agent in the food industries. It forms a gel in the presence of multivalent cations. This gel formation is due to ionic crosslinking of alginate by the replacement of sodium ions with multivalent cations. Thus crosslinked alginate has wide applications in the controlled release of drugs [14,15]. Sterculia gum (SG), also called as karaya gum is a water-soluble high molecular weight (MW ≈ 9,500,000) colloid prepared from the exudates of Sterculia urens tree. It contains three different types of chains; one chain (constituting 50% of the total polysaccharide) contains repeating units of four galacturonic acid residues containing -d-galactose branches and l-rhamnose residues at the reducing end of the unit. A second chain (17% of the polysaccharide) contains an oligorhamnan having d-galacturonic acid branch residues and interrupted occasionally by a d-galactose residues. A third chain (33% of the polysaccharide) contains d-glucuronic acid residues. It constitutes about 13–26% galactose, 15–30% rhamnose and approximately 40% uronic acid residues [16,17]. It is used as a
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bulk laxative [18]. The SG has been reported as a controlled release matrix and has shown better mucoadheshion as compared to guar gum [19]. Repaglinide (RPG) is an oral anti-hyperglycemic drug used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) that acts by stimulating -cells of the pancreas to release insulin. Though RPG shows excellent anti-diabetic activity, it has short half-life (1 h) and low bioavailability (50%). It is rapidly and completely absorbed in the gastrointestinal tract (GIT); however, the absorption is poor from the upper part of the GIT. In addition, it produces hypoglycemia after the oral administration [20]. In view of the fact that this drug is used for long-term treatment, patient compliance is a major concern [21]. The effective control of type II diabetes requires administration of RPG at a dose of 0.5–4 mg three times daily [22]. The drugs, which absorb without difficulty from GIT and have short half-life, are expected to eliminate rapidly from the body and require repeated administration, which may lead to the increased drug toxicity and patient non-compliance. To circumvent this problem, the controlled release systems are being designed to maintain constant drug concentrations in the plasma for longer period [23]. Therefore, the objective of the study was to develop and evaluate dual crosslinked interpenetrating network hydrogel microparticles using SG and SA for controlled release of RPG. The prepared IPN microparticles were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction studies, scanning electron microscopy and evaluated for in vitro drug release behavior and in vivo anti-diabetic activity in wistar rats. 2. Materials and methods
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was emulsified into light liquid paraffin to form water-in-oil (W/O) emulsion using a high speed homogenizer (T 25 Digital Ultra turrax homogenizer, IKA, Germany) in a beaker containing light liquid paraffin and span 80. The solution of CaCl2 or BaCl2 or AlCl3 was added slowly and stirred for 4 h at 500 rpm. The microparticles were filtered, washed with n-hexane and distilled water to remove the oil as well as excess amount of surfactant and they were dried in a hot air oven at 40 ◦ C for 12 h. Further, these ionically crosslinked microparticles were covalently crosslinked by transferring to a methanolic solution containing glutaraldehyde (GA) and 1 N HCl and allowed to react for 30 min at 50 ◦ C. Thus, the dual crosslinked IPN microparticles were washed, dried and stored in a desiccator for further analysis. Formulation composition is shown in Table 1. 2.3. Scanning electron microscopic (SEM) analysis The SEM analysis was performed using required magnification at room temperature. The acceleration voltage used was 10 to 20 kV with the secondary electron image as a detector. The IPN microparticles were coated with platinum to a thickness of about 300 A˚ under an inert atmosphere using a sputter coater module (Edward S 150, UK) in a vacuum evaporator. The coated microparticles were examined under scanning electron microscope (JSM-6360, Jeol, Japan). 2.4. Particle size analysis An optical microscopic method was used to evaluate the size of IPN microparticles. An eyepiece micrometer was used for this purpose, which was calibrated using stage micrometer. Size of 100 particles was measured for each batch and average particle size was calculated.
2.1. Materials 2.5. Drug encapsulation efficiency (DEE) Repaglinide (RPG) was a gifted sample from Torrent Pharmaceuticals (Ahmadabad, India). Sterculia gum (SG), sodium alginate (SA), liquid paraffin (light), span 80, calcium chloride (CaCl2 ), barium chloride (BaCl2 ) and aluminum chloride (AlCl3 ) and glutaraldehyde (GA; 25%) were purchased from HiMedia Laboratories Pvt. Ltd., (Mumbai, India), streptozotocin (STZ) was purchased from CDH Pvt. Ltd. (New Delhi, India). Double distilled water was used throughout the study. All other chemicals were used without further purification. 2.2. Preparation of IPN microparticles
An accurately weighed amount of IPN microparticles were allowed to swell in phosphate buffer of pH 7.4 (100 ml) for 24 h. The swollen microparticles were crushed and the solution was gently heated at 40 ◦ C for 1 h to extract the drug and then centrifuged to remove the polymeric wreckage. The clear supernatant liquid was taken and subjected for drug content estimation using UV–visible spectrophotometer (Model UV-1800, Shimadzu, Japan) at 243 nm. The encapsulation efficiency was calculated using the following equation: DEF =
The IPN microparticles of SG and SA were prepared by emulsioncrosslinking method. The SG and SA polymers were dissolved in double distilled water by stirring to obtain a homogeneous solution. The required amount of RPG was dispersed in polymeric solution and stirred well for 30 min. The drug loaded polymeric solution
Experimental drug content × 100 Theoretical drug content
(1)
2.6. Fourier transform infrared (FTIR) analysis FTIR analysis was performed to study crosslinking reaction and stability of drug within the IPN matrix. The spectra of drug and drug
Table 1 Composition of IPN microparticles. Ingredients
KA1
KA2
KA3
KA4
KA5
KA6
KA7
KA8
Drug (mg) Sterculia gum (% w/v) Sodium alginate (% w/v) Distilled water (ml) Liquid paraffin light (ml) Span-80 (% w/v) CaCl2 (% w/v) BaCl2 (% w/v) AlCl3 (% w/v) Glutaraldehyde (ml) 1 N HCL (ml)
50 1 3 10 100 0.5 8 – – – –
50 2 2 10 100 0.5 8 – – – –
50 3 1 10 100 0.5 8 – – – –
50 2 2 10 100 0.5 – 8 – – –
50 2 2 10 100 0.5 – – 8 – –
50 2 2 10 100 0.5 8 – – 1 1
50 2 2 10 100 0.5 – 8 – 1 1
50 2 2 10 100 0.5 – – 8 1 1
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loaded IPN microparticles were taken on FTIR instrument (FTIR8400S, Shimadzu, Japan) in the range of 4000–500 cm−1 . 2.7. Thermogravimetric analysis (TGA) TG analysis was performed using a microcalorimeter (Diamond TG/DTA, Perkin Elmer, USA) under a dynamic argon atmosphere flowing at a rate of 50 ml/min and at a heating rate of 10 ◦ C/min. in the temperature range 0–400 ◦ C. 2.8. Differential scanning calorimetric (DSC) analysis The DSC analysis of pristine drug, drug loaded IPN microparticles and drug free IPN microparticles was carried out. The thermal analysis was performed by recording thermograms for 5–15 mg samples at a heating rate 10 ◦ C/min in a temperature range 0 ◦ C to 300 ◦ C under argon flow rate of 25 ml/min. using a microcalorimeter (DSC Q20 V24.4 Build 116, TA Instruments, USA). 2.9. X-ray diffraction (XRD) studies The XRD analysis of pristine drug, drug free IPN microparticles and drug loaded IPN microparticles was performed in order to know the crystallinity of drug after formulation. The study was carried out using a benchtop X-ray diffractometer (Rigaku Miniflex II, Japan) in the 2 range 0–80◦ . 2.10. In-vitro drug release behavior The in-vitro release rate of RPG form IPN microparticles was carried out using dissolution apparatus (USP-XXIII Electrolab TDT 08L, Mumbai) consisting 900 ml of buffer of pH 1.2 for the first 2 h and phosphate buffer of pH 7.4 till end of the study at 37 ± 0.5 ◦ C and 100 rpm. Drug release study was carried out for 24 h. The 5 ml aliquots were withdrawn at different time intervals and replaced with an equal amount of fresh dissolution medium. Further, the samples were filtered through a 0.45 mm membrane filter and the amount of drug released was determined using UV–visible spectrophotometer (Model UV-1800, Shimadzu, Japan) following suitable dilutions at 243 nm. 2.11. In-vivo anti-diabetic activity Albino rats of wistar strain (150–250 g) were selected and kept in standard polypropylene cages under controlled room temperature. The rats were fed with standard laboratory diet and water. Animals were kept under fasting for 24 h with water ad libitum during the experiments. Experimental protocol was approved by the institutional animal ethics committee (IAEC), registered under CPCSEA. The rats were made diabetic by intraperitoneal injection of freshly prepared streptozotocin solution at a dose of 60 mg/kg dissolved in water for injection. After 48 h of administration, the rats with fasting blood glucose of 180 mg/dl or more were taken as diabetic and used for further studies. The rats were divided into four groups of six rats each and treated as follows, Group I: normal control rats, Group II: diabetic control rats, Group III: standard rats (treated with pristine repaglanide), Group IV: test rats (treated with KA8 IPN microparticles). The pristine drug and KA8 IPN microparticles were administered orally as suspension in 2% acacia. Blood samples were collected from retro-orbital plexus of each rat under mild anesthesia at 0, 1, 3, 6, 9, 12 and 24th hour after administration and they were analyzed for blood glucose.
Table 2 Average size, drug entrapment efficiency, diffusion coefficients (D) and release parameter (n) of the IPN microparticles. Microparticles
Average size (m)
KA1 KA2 KA3 KA4 KA5 KA6 KA7 KA8
52.12 58.35 61.52 54.56 51.85 55.41 53.25 49.75
± ± ± ± ± ± ± ±
8.13 7.38 3.78 4.25 9.45 9.32 6.42 7.21
DEE (%) 87.83 83.64 81.10 84.59 89.36 85.87 86.71 91.70
± ± ± ± ± ± ± ±
3.88 8.66 7.36 2.16 4.38 4.14 4.32 6.75
D (cm2 /s)
n
6.01 × 10−6 6.21 × 10−6 6.36 × 10−6 5.87 × 10−6 5.23 × 10−6 5.01 × 10−6 4.86 × 10−6 4.23 × 10−6
0.68 0.64 0.61 0.66 0.68 0.73 0.77 0.79
All the values are average of three determinations. ± Indicates SD values. D indicates diffusion coefficient values and n values indicate release mechanism.
2.12. Histopathology of rat pancreas At the end of the study, rats were sacrificed, pancreas were isolated, washed with normal saline and quickly fixed in 10% formalin. The pancreatic tissue was processed according to standard histopathological technique. The tissue was dehydrated in ascending degrees of isopropyl alcohol, cleaned in xylene and impregnated in paraffin wax. The 5 m thick paraffin wax boxes were made, sections were used for cutting microtome and stained with hematoxylin–eosin method and then observed under binocular light microscope (CXRIII, Labomed, India). All the results were expressed as ±SD. Statistical evaluation was done using one-way analysis of variance (ANOVA), followed by Student’s t-test. Differences in mean values were considered significant at p < 0.05. 3. Results and discussion 3.1. Preparation of IPN microparticles The IPN microparticles of SG and SA were prepared by emulsion crosslinking method for the controlled release of RPG. On addition of CaCl2 or BaCl2 or AlCl3 solutions to the polymeric solution of SG and SA, ionic cross-linking occurs between two SA chains and different parts of the same polymer chain. The exchange of Na+ ions occurs with Ca2+ or Ba2+ or Al3+ ions at the carboxylate site and a second strand of SA can also be connected to the same by forming a link in which the counter ions are attached to two or three SA strands jointly. Further, on treating these ionically crosslinked microparticles with GA, an acetal structure has been formed between the CHO groups of GA and OH groups of SA and SG chains to form an IPN including SA and SG, thus making the polymer matrix insoluble (Fig. 1). 3.2. SEM and particle size analysis The shape and surface morphology was examined by SEM analysis; IPN microparticles possess smooth surface as shown in Fig. 2. The average size of IPN microparticles was in the range of 49.75 to 61.52 m (Table 2). Size of the microparticles was increased as the concentration of SA was decreased; the size was also depended upon the type of ionic crosslinking agents. AlCl3 produced smaller microparticles than BaCl2 , which in turn produced smaller microparticles than CaCl2 . During cross-linking, the polymer network might have undergone syneresis resulting in formation of smaller microparticles at higher cross-link densities. Similar results have been reported earlier [24]. As compared to ionically cross-linked microparticles (KA3–KA5), the size of the dual crosslinked microparticles (KA6–KA8) was smaller; it may be due to rapid shrinking of the polymer matrix due to the formation of covalent cross-links
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Fig. 1. Schematic representation of the prepared IPNs.
between the polymer chains resulting in rigid IPN network. This is in agreement with the previously published results [25]. 3.3. Drug encapsulation efficiency DEE of the IPN microparticles was in the range of 81.10% to 91.70% (Table 2). The DEE was decreased with decrease in SA concentration. DEE of the microparticles prepared with Al3+ ions was highest than those prepared with Ba2+ , which in turn was higher than the microparticles prepared with Ca2+ ions. In case of microparticles prepared with Ca2+ ions, the polymer network might be loose with larger pores that results in leakage of entrapped drug into the external medium from polymer matrix during the preparation, leading to lower DEE. Whereas, in case of microparticles prepared with Al3+ ions, the polymer matrix is rigid and leakage
of drug from polymer matrix is low leading to higher DEE. The DEE of microparticles prepared by ionic cross-linking was lower than those prepared by dual cross-linking (both ionic and covalent crosslinking), it may be due to the formation of more stiffer matrix, that reduces the leakage of entrapped drug from the IPN matrix. Similar observations were also reported earlier [26]. 3.4. FTIR analysis The FTIR spectroscopy was used to confirm the crosslinking and IPN formation. Fig. 3 displays the FTIR spectra of SA (A), SG (B) and placebo IPN microparticles KA8 (C), pristine RPG (D) and drug loaded KA8 microparticles (E). In case of SA (A), the broad peak appearing at ∼3400 cm−1 is due to stretching vibrations of hydroxyl groups. The peaks at 1618 and 1683 cm−1 are assigned to
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Fig. 2. Scanning electron microscopic photographs of IPN microparticles (A) and surface morphology (B).
the deformation of carbonyl groups of SA; the peak appearing at 2924 cm−1 is due to the C H stretching of cyclic aldehyde and the peaks appeared at 1031 and 1099 cm−1 are due to C O stretching of alcoholic groups. In the spectra of SG (B), the broad peak appearing at 3425 cm−1 is due to stretching vibrations of hydroxyl groups. The peak appearing at 1630 cm−1 corresponds to deformation of carbonyl group of SG; the peak appearing at 2924 cm−1 is due to the C H stretching of cyclic aldehyde and the peaks appeared at 1043 and 1149 cm−1 are due to the C O stretching of alcoholic groups. While in the spectra of placebo IPN microparticles KA8 (C), the peak at ∼3300 cm−1 is due to stretching vibrations of OH groups of the polysaccharides; sharp peaks appearing at 1687 and 1627 cm−1 correspond to carbonyl functional groups of polysaccharides. The peak at 1438 cm−1 is due to symmetric stretching of the carboxylate groups, whereas the peak appearing at 1036 cm−1 represents the C O C stretching vibrations. The relatively sharp peak appearing at 1215 cm−1 is corresponding to the formation of acetal structures due to reaction between hydroxyl groups of SG & SA and aldehyde groups of GA. During crosslinking, GA reacts with hydroxyl groups of SG and SA in the presence of each other to form interpenetrated polymer network through acetal structures. This could be further supported by the presence of sharp and relatively high intensity peak at 2933 cm−1 due to CH2 groups of the alkyl chain formed by crosslinking [27]. This confirms the crosslinking and IPN formation. FTIR spectroscopy was also used to know the drug stability within IPN matrix. The pristine RPG (D) exhibits peak at 3308 cm−1 is due to stretching of hydroxyl groups. The peak at 2928 cm−1 is due to aliphatic C H stretching, peak at 1687 cm−1 is due to C O stretching of ketone and the peak at 1637 cm−1 is due to carbonyl
Fig. 3. FTIR spectra of SA (A), SG (B) and placebo IPN microparticles KA8 (C), repaglinide (D) and drug loaded KA8 microparticles (E).
groups. Whereas in the spectra of drug loaded KA8 microparticles (E), same characteristic peaks related to pristine RPG were observed with slight deviations. This indicates the stability of drug within IPN matrix. 3.5. TG analysis The typical thermograms of samples are shown in Fig. 4. In case of ionically crosslinked microparticles (KA2, KA4 and KA5), the decomposition of matrix started at lower temperature and we observed a sudden mass loss. KA2 microparticles started decomposing after 100 ◦ C and 24.71% mass loss up to 100 ◦ C is due to loss of loose and bound water in the matrix. A sharp mass loss of 49.08% was observed between 100 ◦ C and 200 ◦ C and it reached a value of 75.32% at 400 ◦ C. This may be attributed to the decomposition of polymer matrix. The decomposition of KA4 and KA5 microparticles started after 100 ◦ C and mass loss (21.67% and 12.38%) up to 100 ◦ C is due to the loss of water present in the matrix. A 45.66% mass loss was observed between 100 ◦ C and 200 ◦ C and it reached a value of 68.42% and 61.19%, respectively, at 400 ◦ C. Thus, the thermal stability of microparticles prepared with AlCl3 was more as compared to those prepared with BaCl2 , which in turn was more thermally stable than the microparticles prepared with CaCl2 . Whereas, in case of dual crosslinked IPN microparticles (KA6, KA7 and KA8), the matrix decomposition started at higher temperature, mass loss was found to be constant and percent residual mass was higher than the ionically crosslinked microparticles. The
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Fig. 6. X-ray diffractograms of pristine repaglinide (A), drug free KA8 microparticles (B), and drug loaded KA8 microparticles (C). Fig. 4. TGA thermograms of KA2 (A), KA4 (B), KA5 (C), KA6 (D), KA7 (E) and KA8 (F) IPN microparticles.
KA6, KA7 and KA8 microparticles have shown mass loss of 59.28%, 51.17% and 40.30%, respectively, at 400 ◦ C. The dual crosslinked microparticles have shown greater thermal stability than the ionically crosslinked microparticles. In case of dual crosslinked IPN microparticles, as the polymeric chains are strongly entangled together, the thermal stability is higher. This suggests the formation of IPN comprising of SG, SA and crosslinking agents. 3.6. DSC analysis The DSC analysis of pristine RPG, drug-free KA8 microparticles and drug-loaded KA8 microparticles was carried out and the results are depicted in Fig. 5. The drug-free IPN microparticles have shown an endothermic peak at 121 ◦ C, whereas, drug-loaded IPN microparticles showed an endothermic peak at 117 ◦ C. Thus, appearance of endothermic peak at lower temperature in case of drug-loaded IPN microparticles may be due to the formation of loose matrix because of creation of extra free space after drug loading. The pristine RPG has shown a sharp endothermic peak at 136 ◦ C due to melting of the drug, but this endothermic peak is not observed in drug-loaded IPN microparticles. This suggests the uniform and amorphous dispersion of drug in the IPN matrix.
Fig. 5. DSC thermograms of pristine repaglinide (A), drug free KA8 microparticles (B) and drug loaded KA8 microparticles (C).
3.7. XRD studies The X-ray diffractograms of pristine RPG (A), drug free KA8 microparticles (B) and drug-loaded KA8 microparticles (C) are presented in Fig. 6. Pristine RPG has shown distinctive intense peaks between the 2 of 12◦ and 25◦ due to crystallinity of drug. Whilst, in case of drug free and drug loaded IPN microparticles, no intense peaks related to drug were noticed between the 2 of 12◦ and 25◦ . But in the diffractogram of drug-loaded KA8 microparticles, few peaks were appeared between the 2 of 25◦ and 30◦ that may be due to surface adhered drug. The diffractograms of both drug-free and drug loaded IPN microparticles are almost one and the same; this indicates the amorphous dispersion of drug in IPN matrix. 3.8. In-vitro drug release studies The in-vitro drug release behavior of IPN microparticles was performed using dissolution rate test apparatus in 0.1 N HCl (pH 1.2) for 2 h and in phosphate buffer (pH 7.4) until end of the study. The drug release profiles of RPG are shown in Fig. 7. Results indicated that the drug release was continued up to 24 h depending upon the formulation variables. The drug release depended upon the types of crosslinking agents; the release was slow from the microparticles, which were prepared with Al3+ as compared to those prepared with Ba2+ ions, which in turn was slower than the microparticles prepared with Ca2+ ions. The GA treatment of these microparticles
Fig. 7. In vitro release profiles of repaglinide from IPN microparticles.
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has further resulted in decreased drug release. As the concentration of SA was decreased, drug release was increased. The ionically crosslinked microparticles discharged the drug quickly while, dual crosslinked IPN microparticles extended the drug release for longer period. This may be due to decrease in free volume of the IPN matrix at higher crosslinking, which obstructs the movement of drugs through the IPN matrix [25]. The diffusion coefficients of drug from IPN microparticles were computed using the following equation [28]:
D=
r 6M∞
2
(2)
while is the slope of linear portion of the plot of Mt /M∞ versus t1/2 , r is radius of microparticles and M∞ is the total quantity of drug loaded. The diffusion coefficients were calculated based on the Fickian diffusion model and the D values are listed in Table 2. The diffusion coefficient of drug was increased with decrease in the concentration of SA. The D values were depended upon the type of crosslinking agent. For example, the D value of KA2 microparticles prepared with Ca2+ ions was higher than KA4 microparticles, which were prepared with Ba2 + ions, and the latter was higher than the KA5 microparticles, which were prepared with Al3+ ions. The Ba2+ ions can produce stronger PIN matrix than Ca2+ ions, whilst Al3+ being a trivalent ion produces still stronger matrix with higher crosslink density than the Ca2+ and Ba2+ ions. Similar results were also reported earlier [24]. Further, D values were lower for the microparticles prepared with dual crosslinking as compared to ionically crosslinked microparticles. It is evident from the study that the increased crosslinking resulted in increased rigidity of IPN network, thus decreasing the drug diffusion. To know the mechanism of drug release, dissolution data was fitted into the following empirical equation [29]: Mt = Kt n M∞
Fig. 8. Blood glucose level of rats treated with pristine repaglinide and repaglinide loaded KA8 IPN microparticles.
(3)
where Mt is quantity of drug released at time t, and M∞ is the total quantity of drug loaded into microparticles, n values indicate the type of release mechanism. The n values are computed for the IPN microparticles and listed in Table 2. The value of n depends upon the type of crosslinking agent; it increases with increased crosslink density. The computed n values indicated that the mechanism of drug release followed non-Fickian transport. Further, KA8 IPN microparticles were selected for in vivo anti-diabetic activity because this formulation has demonstrated satisfactory in vitro drug release behavior. 3.9. In-vivo anti-diabetic activity The pristine RPG and RPG loaded IPN microparticles (KA8) were administered to the streptozotocin induced diabetic rats at a dose of 1.44 mg/kg bw after converting human dose to rat dose. Blood glucose level in diabetic rats were raised nearly 4–5 folds as compared to normal control group rats. The increase in glucose level in diabetic control group was found to be significant (p < 0.05) as compared to normal control group. The rats of normal control group (Group I) have shown blood glucose level in the range of 78.83 to 82.33 mg/dl. Rats of diabetic control group (Group II) have shown blood glucose level in the range of 352.2 to 373.33 mg/dl. Rats of Group III, who received pristine RPG have shown blood glucose level in the range of 127 to 280.8 mg/dl, whereas rats of Group IV, who received RPG loaded KA8 IPN microparticles have shown blood glucose level in the range of 61 to 325.8 mg/dl up to 24 h as shown in Fig. 8. In case of rats treated with pristine RPG, a sudden reduction in blood glucose level was observed up to 3 h; afterwards the blood
Fig. 9. Percentage reduction of blood glucose in rats treated with pristine repaglinide and repaglinide loaded KA8 IPN microparticles.
glucose level was recovered. The percentage reduction of blood glucose was 54.9% at the end of 3 h and gradually it decreased to 18.99% at the end of 24 h. It indicates that the pristine drug has rapidly absorbed, showed action and declined in the plasma; it may be attributed to the shorter biological half-life of RPG. While in case of rats treated with KA8 IPN microparticles, the percentage reduction in blood glucose was slow as compared to pristine RPG up to 3 h; however, percentage glucose reduction was attained above 25% within 1 h and maintained up to 24 h. A 25% reduction in blood glucose is considered to be significant hypoglycemic affect as reported earlier [30]. In our study, the percentage glucose reduction was 34.27% at the end of 3 h, and it was gradually increased to 81.27% at the end of 24 h (Fig. 9). This could be due to the slow release and absorption of RPG over longer period of time [31]. This suggests the controlled release of RPG from IPN microparticles over a longer period as compared to pristine RPG. 3.10. Histopathology of rat pancreas Histopathological evaluation of rat pancreas is shown in Fig. 10 and Table 3. The normal control rats show normal cells in the islets of Langerhans and did not show any pathological anomaly. The diabetic control rats showed higher extent of pathological changes like necrosis of islets, hemorrhages and cellular
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521
Fig. 10. Histopathology of pancreas of normal control rat (A), diabetic control rat (B), rats treated with pristine repaglinide (C) and rats treated with KA8 IPN microparticles (D).
Table 3 Histopathology of rat pancreas. Sr. no.
Parameters
Normal control rats
Diabetic control rats
Repaglinide treated rats
Rats treated with IPN microparticles
1 2
Necrosis Nucleus changes/disappearance of nucleus Karyolysis Size of islets Dilation of large vessels Congestion of large vessels Any other remarks
0 0
2 2
2 2
0 0
0 Normal 0 0 0
1 Reduced 2 2 Islet cell hyperplasia
1 Normal 1 1 Congested vessels
0 Normal 1 0 0
3 4 5 6 7
Scores: 0: nil, 1: mild, 2: moderate, 3: severe, 4: very severe.
infiltration, and reduced dimensions of islets. The rats treated with pristine RPG showed partial reinstatement of normal cellular population of islets. The damage of pancreas in diabetic control rats and partial restoration of ˇ cells was more effectively observed in rats treated with KA8 IPN microparticles. It was found that the KA8 IPN microparticles were more effective than the pristine RPG.
reduction in blood glucose within 3 h and afterwards the percentage reduction in blood glucose was decreased. While in case of rats treated with KA8 IPN microparticles, the percentage reduction in glucose was initially slow as compared to pristine RPG, but it was gradually increased to 81.27% at the end of 24 h. It can be concluded from the study that, the developed IPN microparticles are versatile delivery systems for RPG, which could release the drug in a controlled manner for the effective treatment of diabetes mellitus.
4. Conclusions IPN microparticles of SG and SA were successfully developed by emulsion crosslinking method for the controlled release of RPG. The prepared IPN microparticles were spherical with entrapment efficiency as high as 91%. The ionically crosslinked microparticles discharged the drug quickly, while dual crosslinked microparticles extended the release of drug for 24 h. The anti-diabetic activity indicated that the pristine RPG has shown maximum percentage
Acknowledgements One of the authors (Prof. R V Kulkarni) is grateful to Vision Group on Science & Technology (VGST), Department of Science & Technology, Government of Karnataka, India for financial assistance through major grant K-FIST (Level-II) programme 2012-13. Authors are also thankful to Dr. B.G. Mulimani, Vice Chancellor,
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