International Journal of Biological Macromolecules 70 (2014) 37–43

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Controlled drug delivery attributes of co-polymer micelles and xanthan-O-carboxymethyl hydrogel particles Sabyasachi Maiti ∗ , Susweta Mukherjee Department of Pharmaceutics, Gupta College of Technological Sciences, Ashram More, G.T. Road, Asansol 713301, West Bengal, India

a r t i c l e

i n f o

Article history: Received 9 May 2014 Received in revised form 5 June 2014 Accepted 13 June 2014 Available online 20 June 2014 Keywords: O-Carboxymethylation Xanthan polymer Hydrogel particles Copolymer micelles Dissolution efficiency Hypoglycemia

a b s t r a c t Herein, C16 alkyl chain-grafted-xanthan copolymer was synthesized and characterized. The copolymer self-assembled into nanometer-size spherical micellar structures in water and incorporated ∼100% glibenclamide into its deeper lipophilic confines. The micellar dispersion exhibited negative zeta potential value (−27.6 mV). The copolymer micelles controlled the drug release rate in phosphate buffer solution (pH 6.8) for an extended period. Further incorporation of drug-loaded copolymer micelles into O-carboxymethyl xanthan hydrogel particles slowed the drug release rate in HCl solution (pH 1.2) as well as in phosphate-buffered solution (pH 6.8) (releasing only ∼8% drug in 2 h). The drug release data correlated well with the degree of swelling of the hydrogel particles in different drug release media. Scanning electron microscopy revealed spherical shape of the hydrogel particles (600 ␮m). X-ray diffraction and Fourier transform infrared (FTIR) spectroscopy analyses suggested amorphous encapsulation of the drug and its chemical compatibility with the polymers, respectively. Pharmacodynamic evaluation suggested that the formulations had an immense potential in controlling blood glucose level in animal model over a longer duration. In summary, it was pointed out that the copolymer micelles of glibenclamide, a poor water-soluble anti-diabetic, and their subsequent entrapment into hydrogel particles could be a promising approach in the controlled and effective management of diabetes. © 2014 Published by Elsevier B.V.

1. Introduction Glibenclamide is a second-generation sulfonylurea, used in the treatment of type II diabetes. Its hypoglycemic effect is mainly due to stimulation of insulin release from pancreatic beta cells [1]. Glibenclamide is practically insoluble in water which leads to poor dissolution rate and subsequent decrease in its gastrointestinal absorption. Results of several investigations revealed that the absorption of glibenclamide was limited by its dissolution rate [2–4]. The problem was not only limited to glibenclamide, but also to a considerable number of poorly soluble drugs (about 40%) being discovered today [5]. Thus, the enhancement of their oral bioavailability posed one of the most challenging aspects of drug development. Pharmaceutical scientists attempted a wide range of formulation approaches to improve the dissolution rate of glibenclamide. These included ␤-cyclodextrin complexation [6], solid dispersion [7–11], surfactant micellization [12], micronization [13], and others. However, these gained only a limited degree of

∗ Corresponding author. Tel.: +91 9474119931; fax: +91 341 2314604. E-mail address: [email protected] (S. Maiti). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.015 0141-8130/© 2014 Published by Elsevier B.V.

appreciation in improving its oral bioavailability. Even the use of cosolvents did not prove useful due to the concerns of toxicity and other undesirable side effects [14]. Nowadays, copolymer micelles drew much attention as efficient carriers for compounds, which alone exhibit poor solubility, undesired pharmacokinetics, and low stability in a physiological environment. Polymeric micelles consist of a hydrophobic core stabilized by a corona of hydrophilic polymeric chains exposed to the aqueous environment [15]. From the physicochemical viewpoint, polymeric micelles are more stable toward dilution in biological fluids due to their comparatively lower critical association concentration than that of typical surfactants. Furthermore, they can increase drug bioavailability and retention, since the drug is well protected from possible inactivation under the effect of their biological surroundings [16]. Thorough review of literature suggested that the copolymer investigated so far consisted of poly(ethylene oxide) (also known as polyethylene glycol) as hydrophilic component of the micellar systems. However, a variety of polymers were used as hydrophobic components of the copolymers and these included but not limited to poly(amino acids) [17], poly(␦-valerolactone) [18], poly(d-, l-, and dl-lactic acids) [19], poly(␧-caprolactone) [20,21].

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Few reports on polysaccharide-based copolymer were available in the literature. Notable examples were N-palmitoyl chitosan [22], stearyl chitosan [23], dextran-b-poly(dl-lactide-co-glycolide) [24], stearate-g-dextran [25], N-alkyl-N-dimethyl, and N-alkylN-trimethyl chitosan [26]. The naturally occurring hydrophilic polysaccharides could be an interesting corona-forming material for the copolymers because of their non-toxicity and biodegradability. However, not much emphasis was given to evaluate their potentials as hydrophilic components of amphiphilic copolymers. Xanthan gum is an exopolysaccharide produced by the plantpathogenic bacterium Xanthomonas campestris. It consists of d-glucosyl, d-mannosyl, and d-glucuronyl acid residues and variable proportions of O-acetyl and pyruvyl residues [27]. It is widely used in food, cosmetics, and pharmaceuticals because of its encouraging reports on safety [28]. In this investigation, xanthan gum was used as hydrophilic shell-forming material in the synthesis of copolymer. Hydrogels are three-dimensional hydrophilic polymer networks that can swell in water and hold a large amount of water while maintaining the structure [29,30]. In an earlier report, it was mentioned that xanthan gum could not form hydrogel particles by di- or trivalent metal ion-induced gelation technique. However, its O-carboxymethyl derivative did produce the hydrogel particles with trivalent aluminium metal ions [31]. In another embodiment of this study, glibenclamide was incorporated in copolymer micellar and non-micellar forms and possible crystal changes of the drug, drug–carrier interactions, and in vivo hypoglycemic activity was elucidated. 2. Experimental 2.1. Materials Glibenclamide was a gift from Mylan Labs, R&D, Hyderabad, India. Xanthan gum, aluminum chloride, cetyl alcohol, monochloroacetic acid, and alloxan were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. O-carboxymehtyl xanthan gum (degree of substitution of 1.06) was synthesized in the laboratory by the method reported earlier [31]. N,N-dimethyl formamide (DMF) was purchased from Merck Specialities Pvt. Ltd., Mumbai, India. Thionyl chloride (SOCl2 ) and sodium hydride were obtained from Spectrochem Pvt. Ltd., Mumbai, India. Other reagents were of analytical grade and used as supplied. 2.2. Synthesis of C16 alkyl chain-grafted-xanthan copolymer Cetyl alcohol (3.33%, w/v) was placed in chloroform and SOCl2 (2:1) mixture and refluxed without heat for 2 h for the synthesis of cetyl chloride. Sodium hydride (1.67%, w/v) was added to a dispersion of xanthan gum in DMF (3%, w/v). Cetyl chloride (6.67%, w/v) dissolved in DMF was then transferred to the resulting mixture and stirred for 20 min. Then, the reaction mixture was added to 50 ml of water and adjusted to pH 7.0 using diluted glacial acetic acid. The copolymer was precipitated in ethanol for purification and finally dried. 2.3. Characterization of the copolymer Beilstein test was performed to confirm the synthesis of cetyl chloride. Here, the tip of a copper wire was heated in a burner flame until no further coloration of the flame was noted. The wire was then slightly cooled and dipped into unknown sample. The sample was heated and observed for green flash, indicative of chlorine. Fourier transform infrared (FTIR) spectra of pristine xanthan gum and C16 alkyl chain-grafted-xanthan copolymer were obtained

in Perkin-Elmer IR Spectrometer (Spectrum RX1, UK) using KBr pellets. 2.4. Preparation of drug-loaded micelle Initially, 25 mg of the copolymer was dispersed into 10 ml of deionized water and magnetically agitated to dissolve for 0.5 h, with occasional moderate heating. The required amount of drug (20 mg), dissolved in chloroform (3 ml), was added to the aqueous copolymer solution and stirred for 4 h for complete evaporation of the solvent. Thereafter, the micellar dispersion was filtered by Whatman filter paper (No. 41). 2.5. Characterization of drug-loaded micelle Accurately measured, 4 ml of the filtrate was evaporated to dryness. To this, 10 ml of methanol was added and the absorbance was noted. The drug content was estimated by using the slope of the standard curve of the drug. The recovery analysis averaged 98.26 ± 2.01%. The micellar dispersion was examined under Magnus digital microscope. An optical combination of eye piece (10×) and objective (4×) was used. The photographs were captured by Moticam 1000 camera (Motic, Canada). Malvern Zetasizer Nano ZS 90 apparatus (Malvern Instruments, Worcestershire, UK) equipped with a DTS 1060 cell was used to measure the size and zeta potential value of the micellar sample. The measurements were made in triplicate at 25 ◦ C. 2.6. Preparation of xanthan-O-carboxymethyl hydrogel particles As a first step, 25 mg of copolymer was dispersed in 10 ml of distilled water. Then, 20 mg of glibenclamide was dissolved in chloroform (3 ml) and subsequently added to the copolymer dispersion for drug loading. After 4 h, 75 mg of O-carboxymethyl xanthan was added and stirred to get a homogeneous dispersion. The homogenous dispersion was then added drop-wise through 21-gauze flat tipped needle into 50 ml of 1% (w/v) aluminium chloride (AlCl3 ) solution. The sol droplets were incubated in the gelation medium for 10 min, and the hydrogel particles thus formed were isolated by filtration, washed with distilled water, and air-dried. Xanthan-O-carboxymethyl hydrogel particles containing nonmicellar glibenclamide were prepared as follows. Carboxymethyl xanthan gum (100 mg) was dispersed in 10 ml of water. To this, 20 mg of pure glibenclamide (non-micellar) was added and stirred to obtain a homogenous mixture. This dispersion was added dropwise through 21-gauze flat tipped needle into AlCl3 solution and incubated. The particles were separated and dried. Similarly, the drug-free micellar and non-micellar hydrogel particles were prepared for conducting swelling study in different media. 2.7. Measurement of particle size The diameter of dried hydrogel particles was measured with a digital caliper having an accuracy of 0.01 mm. The diameter of 50 particles from each formulation was measured and averaged. 2.8. Scanning electron microscopy The micellar and non-micellar hydrogel particles were examined under a scanning electron microscope (JEOL-JSM-6360, serial no.: G5/IL/42/08, JEOL Datum Ltd., Japan). Palladium-coated samples were photographed at an acceleration voltage of 17 kV.

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2.9. Drug entrapment efficiency Accurately weighed, 10 mg of beads was digested overnight in 50 ml of phosphate-buffered solution (pH 6.8). The solution was filtered and then 4 ml of the filtrate was evaporated to dryness. The dried material was dissolved in 10 ml of methanol and analyzed for drug content spectrophotometrically at 204 nm. All samples were analyzed in triplicate. The drug entrapment efficiency was calculated by using the ratio of actual drug content to that of theoretical drug content, multiplied by 100.

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fed on 5% glucose solution to prevent hypoglycemia for 24 h and the glucose level was monitored for 7 days until a stable level was achieved. Then, the blood samples were collected from ear vein to ensure diabetic condition. The animals were divided into three groups and each group consisted of five rabbits. Group I received aqueous pure drug dispersion (2.5 mg/kg body weight); Group II and Group III received micellar and non-micellar particles, respectively. The blood samples were collected after oral administration over a period of 8 h and analyzed by Accu-Chek Glucometer Sensor comfort (Roche Diagnostics, Germany) test strips. The percentage reduction in blood glucose level was plotted as a function of time.

2.10. Measurement of swelling ratio Known quantity of drug-free micellar and non-micellar particles (10 mg) were immersed in 10 ml of HCl solution (pH 1.2) or in phosphate-buffered solution (pH 6.8). The swollen particles were removed after 1.5 h, blotted with tissue paper, and weighed. The swelling ratio was calculated by the following the equation: Swelling ratio =

2.14. Data analysis The drug entrapment and dissolution efficiency data were analyzed by one-way analysis of variance (ANOVA): single factor using GraphPad Prism Version 3.00 software. Unpaired t-test was applied to in vivo data. Significant difference was considered at 95% confidence level.

weight of swollen particles − weight of dried particles weight of dried particles 3. Results and discussion

2.11. In vitro drug release testing Accurately weighed, the micellar or non-micellar particles containing equivalent amount of drug was placed in a dialysis bag (MWCO 12-14 kDa) containing 2 ml of phosphate-buffered solution (pH 6.8). The dialysis bag was immersed in phosphate-buffered solution and the volume was adjusted to 900 ml. The paddle of dissolution rate test apparatus (VDA-6D, Veego Instruments Corporation, Mumbai, India) was rotated at 50 rpm. The temperature of the dissolution vessel was maintained at a temperature of 37 ± 0.5 ◦ C. Five milliliters of sample was withdrawn each time and replenished with the same volume of fresh buffer solution. The samples were collected at specified time intervals and analyzed (UV1, Thermo Scientific, UK) at 204 nm. The drug release testing was repeated in HCl solution (pH 1.2) and analyzed at 203 nm. Three samples were tested in each medium. In another study, 2 ml of micellar dispersion was placed in the dialysis bag and immersed in both acidic and alkaline dissolution medium. The rest of the process was similar to that described earlier for the hydrogel particles. 2.12. Infrared spectroscopy and X-ray crystallography Fourier transform infrared (FTIR) spectra of glibenclamide, micellar- and non-micellar hydrogel particles, and drug-free particles were recorded in Perkin-Elmer IR Spectrometer (Spectrum RX1, UK) using KBr pellets. X-ray crystallographic patterns of the samples were traced with X-ray diffractometer (Ultima III, model; D/Max 2200, Rigaku Corporation, Japan) using Cu K␣ radiation at a scan rate of 2◦ min−1 in terms of 2 angle. 2.13. In vivo hypoglycemic effect Non-micellar and micellar hydrogel particles and aqueous drug dispersion containing an equivalent amount of drug were evaluated for their controlled anti-diabetic potential on healthy rabbits (1.2–2.0 kg). This study was conducted with prior approval of Institutional Animal Ethics Committee (Registration No. 955/A/06/CPCSEA). The animals were kept under fasting condition with water ad libitum for 12 h and their normal blood glucose levels were noted. Single IV water injection of alloxan (80 mg/kg body weight) was given to induce experimental diabetes. Rabbits were

3.1. Characterization of C16 alkyl chain-grafted-xanthan copolymer The structure of a hydrophilic xanthan polysaccharide gum chemically modified to design a novel amphiphilic material. The conversion of cetyl alcohol was accomplished successfully. As evidence, a simple qualitative Beilstein test was performed. Cetyl chloride sample indicated a green flash in a burner flame and thus the test was found positive. The synthesis of copolymer was confirmed by FTIR spectra analysis. Besides all the characteristics, stretching vibration peaks due to functional groups present in native xanthan gum and copolymer; a sharp, new peak was evident at 1065.58 cm−1 . This was indicative of C O C stretching of ether group [32] and thus confirmed the synthesis of C16 alkyl chain-grafted-xanthan copolymer (figures not given). The copolymer formed core-shell nanostructure after dispersion of the copolymer in water and this was termed as ‘copolymer micelles’. An insoluble drug glibenclamide was incorporated into micellar structure by solvent evaporation method. The micelles had z-average diameter of 652.8 nm. In general, zeta potential value indicates the stability of any nanocarrier system and a value greater than −25 mV suggests sufficient stability of the system. The present copolymer micellar dispersion showed a negative zeta potential value of −27.6 mV, suggesting its physical stability. This was supported further by microscopic observation in that there was no sign of aggregation of the nanomicellar carriers. The copolymer micelles showed a drug-loading efficiency of 98.75 ± 0.18% (Table 1). The shape of copolymer micelles looked spherical under microscope (Fig. 1a). In a recent study, six novel N-alkyl-N-dimethyl and N-alkyl-Ntrimethyl chitosan derivatives were synthesized. The alkyl groups included octyl (C8 H17 ), decanyl (C10 H21 ), and lauryl (C12 H25 ). These were capable of forming polymeric micelles in water with an average particle diameter ranging from 36 to 218 nm [26]. In another study, block copolymer micelles of dextran and poly(dllactide-co-glycolide) were reported to be spherical and smaller than 100 nm, with a narrow size distribution [24]. Du et al. [25] reported stearate-g-dextran micelles for which the doxorubicin entrapment efficiency reached to up to 84%. Given the literature reports, it was very much clear that the diameter of polymeric micelles was much smaller than the present xanthan copolymer. However, it was interesting to note that the micellar system of this

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Table 1 Preparative conditions and some important properties of the micellar and non-micellar formulations. Code

Carboxymethyl xanthan gum (mg)

Copolymer (mg)

Drug (mg)

Drug entrapment efficiency (%), mean ± SD, n = 3

Size of micelles (nm)/particles (␮m), mean ± SD

F1 F2 F3

– 75 100

25 25 –

20 20 20

98.75 ± 0.18 77.29 ± 0.72 96.66 ± 0.89

652.8 ± 35.62 599.2 ± 72.85 629.2 ± 80.87

present invention was superior with respect to drug entrapment efficiency. 3.2. Characterization of hydrogel particles Inotropic gelation method was used for the preparation of carboxymethyl xanthan hydrogel particles. The gelation was accomplished by ionic interaction of the negatively charged carboxyl function groups with trivalent aluminium metal ions. As soon as the droplets of carboxymethyl xanthan polymer contacted with the ionic crosslinking medium, the droplets turned into insoluble hydrogel particles. The particles had drug entrapment efficiency of 77.29 and 96.66%, respectively, for micellar and non-micellar drugs. The drug entrapment efficiency of the formulations (F1–F3) was statistically different (p < 0.05). It was easy to understand that the drug loading was achieved under aqueous environment. Thus, it was not difficult to assume that the micellar glibenclamide diffused outside the particles due to enhanced aqueous solubility of the drug after micellization and resulted in comparatively lower drug entrapment efficiency. On the contrary, the non-micellar drug was practically insoluble in water and thus its diffusion was hindered by its inherent physical nature from the hydrogel particles. As a consequence, the drug entrapment efficiency of non-micellar

particles was considerably higher (Table 1). It was stated that as gelation proceeds water is expelled due to crosslinks formed by the cations. The dissolved fraction of the drug could be lost from particles during incubation by simple diffusion and the expulsion of water will cause convective loss of the drug molecules [33]. Scanning electron microscopy of both micellar and non-micellar hydrogel particles did not reveal any appreciable differences in their morphological structures. A representative photograph of micellar hydrogel particles was displayed in Fig. 1b. In gelation medium, both the particles appeared spherical at wet state during preparation, but distorted after drying. SEM images did not reveal smoothness of the particle surface (Fig. 1b). The particles had a diameter of 599.2 and 629.2 ␮m, respectively, for micellar and non-micellar particles (Table 1). The particle characteristics were compared with the recent reports on polysaccharide-based hydrogel systems, prepared by this method. Aluminium ion crosslinked polyacrylamide-grafted-xanthan particles had a size range of 960–1003 ␮m and appeared spherical with rough surface. Moreover, a maximum ketoprofen entrapment efficiency of 86.17% was attained [34]. About 83% drug entrapment efficiency was reported for aluminium ion cross-linked carboxymethyl xanthan particles [31]. Aluminium carboxymethyl guar gum beads showed a maximum of 85% drug entrapment efficiency for a water-soluble protein drug; however, their surface appeared to be wrinkled [35]. They further demonstrated that maximum drug entrapment was achieved at much lower concentration of trivalent ions than the divalent metal ions. The particles cross-linked with divalent metal ions showed as low as 35 and 53% entrapment efficiency, respectively, for 0.75 M BaCl2 and CaCl2 concentration. On the other hand, AlCl3 showed 85% entrapment at 0.08 M concentration. It was stated that because of higher valency, it became easier for them to conjugate at least two anionic sites of the sodium carboxymethyl guar gum to effect cross-linking. In the case of divalent ions, it was possible that some of the divalent ions were conjugated with only one of their valences to the polymer, and spatial restrictions did not allow these ions to conjugate with another anionic site on the polymer to effect cross-linking [36]. That was why trivalent metal ion was used for cross-linking of the micellar and non-micellar hydrogel particles.

3.3. Drug release property

Fig. 1. Morphological behaviors of (a) micelles and (b) micelle-loaded hydrogel particles.

The drug release curves of the formulations (F1–F3) in phosphate-buffered solution were given in Fig. 2. The micellar dispersion released about 35 and 82% incorporated drug in 2 h and 8 h, respectively. It was said that the micelles not only incorporated a greater amount of drug, but also exhibited potential to act as controlled release drug carriers. The possible causes for the enhanced dissolution of drug from the micellar dispersion could be increased wettability of the drug by the carrier, nanometer size of the micelles, and polymorphic transformation of drug crystals [37]. Incorporation of the drug either in micellar or non-micellar form into hydrogel particles decreased the drug release rate to a significant level, releasing only 22–27% drug at the end of 8 h. However, the release profile of micellar particles rose slightly above to that of the micellar particles. The dissolution testing was conducted in the dialysis bag. In the case of micellar preparation (F1), the drug

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Fig. 2. In vitro drug release curves of different formulations in phosphate-buffered solution (pH 6.8). Key: () F1; () F2; (䊉) F3.

release inside the bag permeated the bag and underwent solution into outside dissolution medium and provided a faster drug release profile. When incorporated into the hydrogel particles, the micellar drug released first into the fluid retained inside the bag, then liberated the drug, and lastly permeated via membrane to outside dissolution medium. In this way, the drug release was hampered and the initial release rate delayed due to delay in hydration and consequently swelling of the particles. On the contrary, the non-micellar drug diffused after swelling of the particles ensured solution immediately and then diffused outside the bag. The micellar drug followed two steps, i.e., release of micellar drug into dialysis bag fluid and then release of drug from micelles into dialysis bag fluid. This complex phenomenon could be responsible for demonstrating such release behavior. The drug release property was also evaluated under simultaneous pH environment as found in vivo (Fig. 3). Only 22% drug released into acidic medium after 2 h from micellar formulation (F1) and reached up to 83% thereafter at the end of 8 h. A maximum of 7–8% drug ensured solution in 2 h from the micellar and non-micellar hydrogel particles. Thus, the drug release was comparatively slower in acidic medium. This was attributed to lower swelling of the particles in acidic medium (Table 2). It might so happen that the carboxyl group of carboxymethyl xanthan gum remained in ionized form in higher pH values and a repulsive force created inside the beads and thus swelled more rapidly. The opposite phenomenon could be expected in acidic medium. Further, the drug release was also found to be improved after simultaneous dissolution study of the micellar and non-micellar particles at the end of 8 h. However, the trend in drug release did not change in the case of micellar and non-micellar particles. In addition to percentage drug release data, the dissolution efficiency was considered as a parameter for comparison of the drug release profiles between the formulations. A model-independent

Fig. 4. FTIR spectra of (a) pure drug; (b) blank hydrogel particles; (c) non-micellar hydrogel particles; and (d) micellar hydrogel particles.

parameter, the dissolution efficiency, DE (%), was employed to compare the dissolution profiles of different formulations [38]:

t DE (%) =

0

yt dt

y100 t

where yt is the percent of drug dissolved at any time t, y100 denotes 100% dissolution, and the integral represents the area under dissolution curve between time zero and t. The dissolution efficiency (DE8 %) data obtained after dissolution in phosphate-buffered solution in 8 h was presented in Table 2. There was no significant difference between the DE8 (%) data as was noted statistically after ANOVA analysis followed by Tukey’s posttest. This post-test was mainly attempted to differentiate between the dissolution curves of the micellar and non-micellar hydrogel particles. Though their profiles seemed superimposable but statistically, these were significantly different (p < 0.001). The drug release data were fitted into Korsmeyer–Peppas model [39]: Mt /M∞ = k tn , where Mt /M∞ is the fractional solute release at time t and k is a constant characteristics of the device. Usually, the value of n between 0.43 and 0.85 indicated anomalous (nonFickian) diffusion. The values of diffusion coefficient were given in Table 2. Simple diffusion mechanism was involved in the case of F1 and F3 formulations (n < 0.43). However, the micellar particles (F2) followed anomalous diffusion mechanism for drug release where the mechanism was coupled by simple diffusion and polymer relaxation. 3.4. Compatibility characteristics

Fig. 3. In vitro drug release curves of different formulations in acid solution (pH 1.2) for 2 h followed by phosphate-buffered solution (pH 6.8) for 6 h. Key: () F1; () F2; (䊉) F3.

In FTIR spectrum of pure drug, the peaks at 1342.01 and 1306.16 cm−1 were attributed to the strong asymmetric S O stretching of the secondary SO2 NH group. The strong symmetric S O stretching of secondary SO2 NH was noted at 1182.25 and 1159.34 cm−1 . The N H stretching of R NH R and C O NH R was noted at 3368.25 cm−1 [11]. Further, the peak at 1715.65 cm−1 indicated C O stretching vibration and NH deformation of C O NH R [40]. The presence of aryl C Cl stretching was found at 400–500 cm−1 (Fig. 4a).

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Table 2 In vitro drug release properties and modeling of drug release data for elucidation of release mechanism. Code

F1 F2 F3 a

Swelling index in HCl solution

– 0.85 1.20

Swelling index in buffer solution

– 2.47 3.21

% drug release in HCl solution

22.14 ± 1.53 7.63 ± 1.21 8.72 ± 1.14

Dissolution efficiency (DE8 %), mean ± SD, n = 3

47.75 ± 0.53 13.32 ± 0.17 17.61 ± 0.01

Korsmeyer–Peppas model parameters k

n

r2 a

0.0061 0.0055 0.0196

0.3840 0.5877 0.4126

0.9156 0.9757 0.9818

Correlation coefficient.

Fig. 5. X-ray diffraction patterns of (a) pure drug; (b) hydrogel particles with nonmicellar glibenclamide; (c) hydrogel particles with micellar glibenclamide; (d) drugfree hydrogel particles.

No such peaks were evident in infrared spectra of blank hydrogel particles (Fig. 4b). Similar peaks were observed in the FTIR spectra of micellar and non-micellar hydrogel particles with insignificant changes in wavenumbers (Fig. 4c and d). Hence, it was said that there was no interaction between the drug and the polymer. The pure drug exhibited numerous peaks at 2 of 10.78◦ , 11.64◦ , 18.82◦ , 19.36◦ , 20.88◦ , 23.08◦ , and 27.58◦ in its X-ray diffraction pattern (Fig. 5a) [41]. The characteristic peaks of glibenclamide nearly completely disappeared from the X-ray diffraction patterns of the rest of the sample formulation (Fig. 5b–d). This indicated the formation of an amorphous state of glibenclamide while incorporated. 3.5. Hypoglycemic activity In vivo hypoglycemic activity of the formulations was represented in Fig. 6. The pure drug dispersion reduced a maximum of 30.19% glucose level at the 4th hour and the curve started declining to 20.23% at the end of 8 h.

Fig. 6. In vivo pharmacodynamic activity of the formulations on lowering of blood glucose level in rabbit models. Key: (♦) pure drug; () micellar formulation; () non-micellar hydrogel particles; and (䊉) micellar hydrogel particles.

On the contrary, micellar dispersion exhibited its significant glucose-lowering activity from beginning (0.5 h) and reached a maximum level of 56.59% at the 5th hour and then tended to recover to their normal level. It was very much clear that micellar dispersion provided improved pharmacodynamic activity. This could be attributed to the enhanced solubility of the drug in micellar form as well as their in vivo uptake via biological membranes into the blood stream. Micelles controlled the drug release and thus a prolonged glucose-lowering profile was obtained in vivo. Unpaired t-test revealed a significant difference between the glucose lowering data of the pure drug and micellar dispersion (p < 0.05). With respect to dissolution behavior of the hydrogel particles, the in vivo effect was found contradictory in that the micellar hydrogel particles showed better hypoglycemic activity than the non-micellar hydrogel particles, in spite of their opposite drug release behavior in vitro. Both formulations continued their glucose-lowering action up to 8 h, reducing about 33–37% blood glucose level. The only difference seen was the matter of significant reduction of blood glucose level. Kahn and Shechter [42] suggested that a 25% drop in blood glucose level could be considered a significant hypoglycemic effect. The non-micellar hydrogel particles caused a significant hypoglycemic effect only at the 5th hour, whereas the micellar hydrogel particles did that 1 h earlier. It may so happen that under in vivo conditions, as soon as the micelles liberated from the hydrogel particles, some portions permeated via biological membrane intact due to their nanometer size and reached the systemic circulation and released their content directly in the blood with simultaneous absorption of drug particles from the GI tract. On the other hand, the drug absorption was solely controlled by dissolution rate of the release pure drug. Unpaired t-test, however, did not indicate any significant difference between the blood glucose-lowering activity between micellar and non-micellar particles (p > 0.05).

4. Conclusion Xanthan gum was conferred amphiphilic character after grafting a long C16 lipophilic chain to its backbone. The copolymer was able to form spherical, stable core-shell nanomicellar structures in water and incorporated almost 100% drug in its core as was evident by solvent evaporation technique. The copolymer could enhance the solubility of the drug and prolonged the drug release under in vivo pH conditions. The hydrogel particles of micellar and non-micellar drug retarded the drug release further. The drug was compatible with the copolymer and carboxymethyl xanthan polymer. The result of in vivo hypoglycemic activity of the micellar dispersion and its hydrogel formulation was found promising. In conclusion, this newly designed device had intense potential in controlling glucose level for the diabetic patients.

Conflict of interest The authors report no conflict of interest.

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