The optimization of the formulation was carried out by 32 full factorial designs. The evaluation of two different factors at three different levels was done. The two factors selected as independent variable here are amount of polymer and cross-linking agent. These two factors are varied, and factor levels were suitably coded as − 1, 0, and + 1, respectively, for low, intermediate and high. The responses like drug loading (%), bioadhesiveness (%) and drug release (%) after 12 h were considered as dependant variable. In this design approach, evaluation of two factors was carried out at three levels each and performing the experimental trails for all the combinations possible. Any other processing and also the formulation variables were kept constant throughout the entire study. In total, 9 runs (formulations) were designed by using the software Design Expert version 12. (Stat-Ease Minneapolis, MN, USA). Accordingly, the dependent and the independent variable’s relationship pattern were studied and the analysis of the same was carried out on gaining the surface response to attain the significant model.
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1.
Independent levels:
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2.
Polymer (X1)
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3.
Cross-linking agent (X2)
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4.
Dependent levels:
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5.
Drug Loading (Y1)
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6.
Bioadhesiveness (Y2)
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7.
Drug Release (Y3)
Statistical analysis
The statistical optimization was performed using Design Expert version 12. (Stat-Ease Minneapolis, MN, USA). All measured data are expressed as mean ± standard deviation (S.D.). Each of the measurement was done in triplicate (n = 3).
HPLC analysis of Gliclazide
The chromatographic analysis was carried out on Thermo Scientific (Dionex Ultra 3000plus) HPLC using a stainless steel column (25 cm × 4 mm) packed with end capped octylsilane bonded to porous silica (4 µm) in isocratic elution mode using phosphate buffer pH 3.4 and HPLC grade acetonitrile in the ratio of 20:80 (v/v) as eluent. An aliquot of 20 µl sample was injected to the column by maintaining the flow rate at 1 ml/min. The eluent was detected at 230 nm. [21]
Drug and excipient compatibility study
Fourier transform infrared spectroscopy (FTIR)
To find out any possible molecular interaction among the drug and the excipients used, the Fourier transform infrared (FTIR) spectroscopy (Bruker) was used. The technique utilizes the potassium bromide (KBr) disc method that involves placing of the disc in the sample holder and scanning it from 4000 to 400 cm−1 with the resolution maintained at 4 cm−1. The analysis was carried out for the pure drug, the polymers used and also the formulation loaded with the drug.
Differential scanning calorimetry (DSC)
To estimate out the thermal nature of the pure drug, excipient, excipient mixture along with the gliclazide-loaded microsphere, the technique of differential scanning calorimetry was used. All the samples were allowed to run in a differential scanning calorimeter (JADE, Perkin Elmer, USA,) at the scanning rate of 10°c/min with the measurements conducted by maintaining the heating range of 40–250 °C utilizing nitrogen as the purging gas. Furthermore, the evaluations of any possible interactions in the thermograms were carried out using Pyris software (PerkinElmer, Inc., Waltham, MA, USA). [22]
Isothermal stress testing (IST)
In IST studies, the drug along with different excipients used was weighed directly into a previously cleaned 4 ml glass vials (n = 2) and was subjected to homogenization on a vortex mixture for around 2 min following addition of about 10% water in each of the vials. The drug-excipient blend was further subjected to mixing with the aid of a glass capillary whose both ends were heat sealed. The capillary was broken in order to prevent any wastage of materials, and finally, each of the vials was sealed using a screw cap lined with Teflon and stored by maintaining a temperature of 50 °C in a hot air oven. At periodic time interval, the vials containing the samples were visually inspected for any alteration in colour. The analysis of the sample after storing for 3 weeks was carried out by using UV visible spectrophotometer [23]. A vial containing drug excipient mixture stored in a refrigerator without any addition of water to it served as the control sample for the entire analysis. The sample for analysis purpose was prepared by adding 2 ml of methanol to all the vials. The entire mixture was subjected to homogenization and was transferred to a 100 ml volumetric flask. The vials were further rinsed with methanol twice, and the final volume was made up to the mark. Finally, the samples were centrifuged and the superannuated liquid was filtered through a nylon filter and analysis was carried out for estimating the drug content after proper dilution with the same solvent. In addition, the spectral analysis by peak matching technique was also performed which gives an indication about the change in wavelength maxima in case of the pure drug as well as the drug excipient mixture carried out for both the control as well as the stressed sample.
Characterization of prepared microsphere
Production yield
The yield of production for the microsphere prepared was calculated by determining the weight of the final dried microsphere in context to the total amount of the drug and the polymer used for the preparation. Finally, the percentage of production yield was calculated by using the formula mentioned below
$${\text{Production Yield }} = \frac{{{\text{Mass }}\left( {{\text{Microsphere}}} \right)}}{{{\text{Theoretical Mass }}\left( {{\text{Polymer }} + {\text{ Drug}}} \right)}} \times {1}00$$
Bulk density
The estimation of Bulk Density of the prepared microsphere was carried out by accurately weighing out microsphere (Wm) and then transferring it into a graduated cylinder of 100 ml volume in order to attain the apparent volume (V) in between 50 and 100 ml. The following formula was used in estimating out bulk density in gram/millilitre
$${\text{Bulk density }}\left( {\tilde{n}0} \right) \, = \, \frac{M}{{V_{{\text{o}}} }}$$
where M = powder mass, Vo = powder volume.
Angle of repose
This is a common technique for estimation of the flow property of the prepared microsphere which was carried out by using the fixed funnel method. In this current method, a funnel is used and is fixed to a stand in a way such that its tip achieves a height of 6 cm when measured from the surface. A sufficient amount of microsphere was then allowed to pass through the funnel so that if forms a pile. Finally, the angle of repose was estimated by measuring out the radius and height of the heap of the pile and putting the values in the formula given below.
$${\text{Tan }}\theta = \, \frac{H}{R}$$
Here, H = height of the pile from the surface, R = radius of the pile.
Drug loading and entrapment efficiency
Microsphere equivalent to 100 mg was weighed out accurately and transferred to a mortar and pestle and crushed. To the above crushed powdered microsphere, 500 ml of phosphate buffer solution (PBS) having pH of 7.4 was added and was kept aside for 48 h with occasional shaking. The temperature condition for this study was maintained at 37 ± 0.5 °C. After the specified time, the mixture was filtered to remove the polymer debris generated as a result of disintegration of the microcapsules. The estimation of the drug content was carried out using a double beam Ultraviolet-VIS spectrophotometer (Shimadzu, Japan) maintaining the wavelength at 227 nm. The following formula was used for estimating the % DEE [24].
$${\text{DEE}}\% = \frac{{\text{Actual drug content in microcapsules}}}{{\text{Theoretical drug content in microcapsules}}} \times {1}00$$
Swelling characteristics of microspheres
The swelling behaviour of microspheres of MSB-Starch alginate loaded with gliclazide was studied in two different media. The current study was done in 0.1 N HCl of pH 1.2 and PBS of pH 7.4. This methodology involves the addition of 100 mg of microspheres to the bowl of the USP dissolution apparatus. Following the addition of microspheres, the respective media of about 500 ml was added, and the microspheres were allowed to swell. The temperature condition of the apparatus was fixed at 37 ± 0.5 °C with a paddle speed of 50 rpm [24]. The estimation of swelling behaviour was done by removing the swollen microspheres at regular intervals, then drying the surface with tissue paper and weighing it. The following formula was utilized to calculate the swelling index of the above microspheres maintained in two different mediums.
$$\% {\text{Swelling = }}\frac{{{\text{Weight of Microspheres after Swelling}} - {\text{Weight of Dry Micropheres}}}}{{\text{Weight of dry Micropheres}}} \times {1}00$$
Bioadhesion study
The mucoadhesive nature of the prepared microsphere was assessed utilizing the in vitro wash off technique. The above study involves the utilization of freshly collected and excised intestinal mucosa of goat having dimensions of 2 × 2 cm. This excise piece of intestinal mucosa that was collected from a local slaughter house was than subjected to mounting on a clean glass slide (7.5 × 2.5 cm) by attachment with a thread. The study involves the spreading of about 100 microspheres gently on to the wet and ringed specimen of tissue assembled on to a disintegration test apparatus to allow the continuous up and down movement. The entire study was carried out by using 900 ml PBS solution of pH 1.2 with temperature maintained at 37 ± 0.5 °C. At multiple time intervals, the count for the number of microspheres still attaching to the tissue specimen was done [24].
Surface morphology by Field Emission Scanning Electron Microscopy (FESEM)
The investigation of the morphology of the prepared microsphere was carried out by utilizing scanning electron microscopy (FESEM-S 4800, Hitachi, Japan) by maintaining the working distance of 8.6–8.8 mm and also keeping the accelerating voltage of 1.0 kV. The prepared microspheres were routinely examined for its shape, size and its surface characteristics. [25]
In vitro release study
The amount of drug released from the prepared microsphere was studied using an eight-station paddle type (USP Type II) dissolution apparatus (Lab India) each consisting of 900 ml PBS solution of pH 7.4 with the bath temperature and stirring speed maintained at 37 ± 1 °C and 50 rpm, respectively. It is worth to be mentioned that gliclazide degrades in the gastric pH (1.2) and shows to be more soluble in the alkaline pH value as reported by Bansal et al. [26]. However, the drug remains in the stomach for only 2 h; duration of time the drug proves to be stable. Consequently, the dissolution medium was chosen as PBS solution pH 7.4 as documented in BP 2013. An equivalent to 100 mg of microsphere was taken and added to the six bowls of the dissolution test apparatus, whereas the remaining two bowls were left alone with only the buffer solution as the replacing fluid. At specific time interval, approximately 5 ml of aliquot was collected and subsequently the same volume was replaced with fresh buffer in order to maintain the sink condition thought the entire experimental process. The collected aliquots were filtered and diluted, and estimation for GLZ content was done by using a UV–VIS double beam spectrophotometer (Shimadzu, Japan) at 227 nm wavelength [27].
Kinetics of drug release
The release kinetics serves as a useful tool in correlating both the response obtained from in vitro as well as in vivo analysis. This correlation is done mostly by comparing the pharmacokinetics results with that of the dissolution profile of the formulations. There are several available mathematical models like zero order, first order, Higuchi that helps in predicting the kinetics of drug release. To study the release kinetics of GLZ, DD solver (2010), an add-in program for Microsoft excels used for the purpose of modelling and comparison of drug release profile was used. [28]
Drug release mechanism
For evaluation of the drug release mechanism from the prepared microspheres, the data obtained from the in vitro drug release was plotted in Korsmeyer–Peppas equation [29] by taking log cumulative percent of drug release on Y-axis and log time on X-axis. The exponent “n” was calculated through the slope of the straight line. The equation is
$${\text{M}}_{{\text{t}}} /{\text{M}}_{\infty } = {\text{ Kt}}^{{\text{n}}}$$
Here, “Mt/M∞” indicates the fractional solute release, “t” is the indication of time taken to release and the value of “K” gives a representation of the kinetic constant characteristics in the drug/polymer system. The mechanism of drug release is mostly characterized by the exponent “n” in the equation, and its value is significant in predicting the drug release pattern. If the exponent “n” has a value that is 0.5 or less, the release mechanism mostly follows the Fickian diffusion. Similarly, if 0.5 < n < 1, the release mechanism mostly follows the non-Fickian or the anomalous diffusion. However, in case the exponent n has a value greater than 1, it mostly indicates the release pattern to be super case-II transport mechanism.
In vivo testing of the optimized formulation
The evaluation of the optimized batch of the prepared mucoadhesive microsphere was carried out by adopting several in vivo tests. The evaluation process included the measurement of pharmacokinetic parameters after single oral dose administration, and also pharmacodynamics evaluation of the prepared microsphere on streptozotocin-induced diabetic rats. Moreover, for the assessment of gastro-retentive behaviour, the technique of gamma scintigraphy was employed.
Experimental animal selection and ethical consideration
Healthy New Zealand white rabbits (weighing from 1.5–2 kg) and albino Wistar rats (weighing from 150 to 200 g) of either sex were procured from M/S Chakraborty Enterprise, Kolkata, West Bengal, India, and were used for the assessment of pharmacokinetics study, in vivo gastro-retentive behaviour and antidiabetic study. All the animals were housed in poly-acrylic cages having an assembly of wire mesh at the top along with a hygienic bed of husk in a specified animal room free from pathogen by maintaining the standard laboratory conditions (temperature 25 ± 2 °C and relative humidity maintained at 50 ± 10%) with light/ dark cycle (12/12 h) allowing free access to standard rat feed (VRK Nutritional Solution, Pune, India) and water. The study was approved (approval number- NIPS/AH/20/19) by the Institutional Animal Ethical Committee (IAEC) of NETES Institute of Pharmaceutical Science (NIPS), Kamrup, Assam, India, and was conducted in accordance with the committee for the purpose of control and supervision of experiments on animals (CPCSEA).
Pharmacokinetic study
For the pharmacokinetic study, the overnight fasten rats were divided into two groups with 5 in each (n = 5) in a parallel design. The first group was administered with the optimized batch of microsphere, whereas the second group received raw gliclazide powder all in a dose corresponding to 10 mg/kg gliclazide as such dose was previously reported in the literature. [30]
The doses were dispersed in 2 ml of distilled water and were vortexed for 10 s not more than 1 min before oral administration. This was directly followed by adding 1 ml of glucose solution in a dose of 1 g/kg in order to prevent severe hypoglycaemia that might arise due to gliclazide administration.
A sample of 0.5 mL blood was collected from every rat into an EDTA tube by retro-orbital vein puncture at the following intervals: 0 (pre-dose), 0.5, 1, 2, 3, 4, 6 and 24 h.
Preparation of plasma samples
The collected blood samples were centrifuged at 8000 rpm for 20 min in order to separate out the plasma. The plasma that was separated was collected and stored at − 20 °C for further analysis purpose. The working standard phenytoin (100 µL) having a concentration of 12 µg/ml was added to 100 µl of plasma followed by vortexing for 30 s and continuing centrifugation at 10,000 rpm for 15 min at 20 °C to allow plasma protein separation. Finally, the supernatant was filtered using a 0.2 µm syringe filter and was injected into HPLC system. For each of the sample, GLZ/PHY peak area ratio was recorded and the concentration of GLZ was determined using the calibration curve constructed in plasma. [30]
HPLC analysis of GLZ in rat plasmas
The estimation of GLZ concentration in rat plasma was carried out using the same HPLC method as used in the in vitro quantification by using phenytoin (PHY) as the internal standard.
Pharmacokinetic parameters
The obtained data were further analysed for pharmacokinetic parameters by using PK solver (2010), an add-in program for Microsoft Excel used in analysis of pharmacokinetic and pharmacodynamics data. The area under the plasma concentration time profile (AUC0–24 and AUC0-inf) was calculated using the linear trapezoidal method. Also, the maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (Tmax) were also determined subsequently [30].
Pharmacodynamics study protocol
In vivo anti diabetic study
To access the antidiabetic activity of the prepared mucoadhesive microsphere, streptozotocin (STZ)-induced diabetic rat model was used [30]. For the study purpose, diabetes was induced in the rats by intraperitoneal injection of freshly prepared streptozotocin (STZ) at a dose of 50 mg/kg body weight [31] dissolved in 0.1 M cold sodium citrate buffer solution of pH 4.5. In order to avoid fatal hypoglycemia following administration of STZ, the animals were permitted to drink 5% (w/v) glucose solution for the next 24 h. After 3 days of STZ administration, a drop of blood was collected from tail vein from fasted rats for estimation of blood glucose level (Accu-Chek Extra Care Roche Diabetes Care India Pvt. Ltd.). The animals with serum glucose level more than 250 mg/dl were measured as diabetic and considered for further study. The rats were randomly divided into four groups with six in each (n = 6) as mentioned below.
Group I: Normal control rats (NC); received intraperitoneal injection of 0.9% w/v NaCl solution (1 ml/kg b.w. i.p.).
Group II: Diabetogenic control (STZ); received a single dose of streptozotocin (STZ).
Group III: Standard treatment (STZ + GL); following the STZ injection, this group received Glibenclamide at a dose of 5 mg/kg [32] b.w., p.o for the next three weeks.
Group IV: Test compound (STZ + GLM); alike to group III except that the test drug Gliclazide-loaded microsphere (10 mg/kg b.w., p.o) [33] replaced the Glibenclamide.
The blood glucose level was estimated in all groups, before and after treatment on 3rd, 10th, 17th and 24th day from the overnight fasted rats. Blood samples were collected by retro-orbital venous plexus puncture method under mild ether anaesthesia. Blood was allowed to stand for 10 min at room temperature, centrifuged at 2500 rpm for 10 min to separate the serum for assessment of lipid profile (Triglycerides, Total Cholesterol, High-density lipoprotein cholesterol, low-density lipoprotein cholesterol). All the biochemical parameters were measured using standard commercial kits with the help of a semi-auto-analyser (Erba Chem 7).
All the animals (rabbits and rats) used in both the studies were not subjected to euthanasia since none of the animals was killed after completion of the experimental period. However, post-experimental and after a definite wash out period all the animals were further used for monitoring behavioural changes.
Pharmacoscintigraphic study
To estimate the in vivo gastro-retentive behaviour of the prepared mucoadhesive gliclazide microsphere following oral administration and determine its extent of transit through the GI tract, the gamma scintigraphy technique was used [34]. The overnight fastened rabbits were randomly divided into two groups with 3 in each (n = 3) with one serving as the control group. The radio labelling technique for the prepared microsphere was carried out in a stannous chloride that is a potent reducing agent. This radio labelling technique with 99mTc-pertechnetate involves 3 mg of the gliclazide-loaded mucoadhesive microsphere in a glass vial, which was previously sterilized following the addition of 1 ml of distilled water to it. This was followed by the addition of reducing agents of concentrations about 60 µg and maintaining the pH of the solution to 7.5. Finally, filtration was carried out for the above contents through a Whatman filter paper (#41) into a sterile vial, and an approximate of about 18.5 MBq 99mTc-pertechnetate was added, mixed and incubated for about 5–10 min. Furthermore, to determine the radio labelling efficiency of the complex, the technique of paper chromatography was used. The mobile phase used in this process was acetone (100%). The spotting of the sample was done initially after labelling the microsphere and before the washing step. The pertechnetate tends to migrate to the crest of the stationary phase, whereas the microsphere-attached material tends to remain at the initial point of application. The labelling yield here was expressed as a percentage of the total amount of radioactivity applied in the testing system, calculated using the formula.
$${\text{Radio labelling}} = \, \frac{{{\text{Radioactivity }}\left( {{\text{Counts}}} \right){\text{ Retain in the Lower Half of strip}}}}{{{\text{Initial Radioactivity }}\left( {\text{Total Count Present}} \right){\text{ with the Strip}}}} \times { 1}00$$
To restrict the movement of the animals during imaging, the four legs of the animals were tied to plywood. The radio-labelled formulation was finally administered to the animal of the first group via a feeding tube followed by drinking water in sufficient volume through the same tube. The animals were subjected to imaging with the help of a gamma camera (Siemens AG, Munich, Germany) to locate the formulation. Scanning of the whole anterior body of the rabbit was carried out for 15 min, and the 140 keV gamma rays that were emitted by 99mTc were imaged. The gamma camera used single photon emission computed tomography (SPECT) to detect the amount of emission. The gamma images generated were recorded by utilization of an online computer system.
Statistical analysis
The determination of statistical significance was carried out using the statistical software package Graph Pad Prism version 6.0 (Graph Pad Software, San Diego, CA, USA). The results are expressed as mean ± SEM, and the results were analysed using a one-way ANOVA test. Further, the Dunnett’s multiple comparison tests was followed considering the p values of < 0.05 as significant.