Journal of Drug Design and Research

Chitosan Based Microspheres of Glibenclamide Incorporated in in-situ Gel for Nasal Delivery: Optimization and in-vitro Evaluation

Research Article | Open Access Volume 4 | Issue 1 |

  • 1. Department of Pharmaceutics, Anjali College of Pharmacy and Science, India
  • 2. Department of Pharmaceutics, MM University Mullana, India
+ Show More - Show Less
Corresponding Authors
Manish Kumar, Department of Pharmaceutics, MM University Mullana, India, Tel: 9719021160

The purpose of research work was to develop and optimize mucoadhesive microspheres of glibenclamide incorporated in in-situ gel for nasal delivery with the aim to enhance the residence time and improve therapeutic efficacy. Chitosan (mucoadhesive) based microspheres of glibenclamide were prepared by emulsificationcrosslinking method. Glutaraldehyde was used as crosslinking agent. A 32 factorial design was employed for preparation of microspheres wherein the concentration of polymer and crosslinking agent were selected as independent variables while particle size of microspheres, in vitro mucoadhesion and % cumulative drug permeation were the dependent variables. Microspheres were evaluated with respect to the production yield, particle size, entrapment efficiency, swelling property, in vitro mucoadhesion and % cumulative drug release. Formulation F8 was found to be optimized. The optimized formulation F8 was then formulated into thermo reversible in-situ gel. The prepared micro particulate in-situ nasal gel was mucoadhesive in nature which adhere onto the mucus and increase the residence time within the nasal cavity.


Singh N, Kumar M, Bhatt S, Malik A, Saini V (2017) Chitosan Based Microspheres of Glibenclamide Incorporated in in-situ Gel for Nasal Delivery: Optimization and in-vitro Evaluation. J Drug Des Res 4(1): 1034.


•    Glibenclamide
•    Mucoadhesive microspheres
•    32 Full factorial design
•    Emulsification crosslinking method


The nose is considered as an attractive route for needlefree vaccination and for systemic drug delivery, especially when rapid absorption and effect are desired. In addition, nasal delivery may help address issues related to poor bioavailability, slow absorption, drug degradation, and adverse events in the gastrointestinal tract and avoids the first-pass metabolism in the liver [1]. However, when considering nasal delivery devices and mechanisms, it is important to keep in mind that the prime purpose of the nasal airway is to protect the delicate lungs from hazardous exposures, not to serve as a delivery route for drugs and vaccines [2]. Nasal cavity possesses various advantages as a site for drug delivery, such as it provides much vascularized epithelium, large surface area for drug absorption, lower enzymatic activity compared with the gastrointestinal tract and liver and the direct drug transport into the systemic circulation, thereby avoiding hepatic first pass metabolism and irritation of gastrointestinal membrane [3]. Nasal route is non-invasive, therefore it reduces the risk of infection. Furthermore, ease of use and self-medication possibility result in improved patient compliance [3]. The range of compounds investigated for possible nasal application greatly from very lipophilic drugs to polar, hydrophilic molecules including peptides and proteins. The nasal dosage forms involved include solutions, sprays, gels, liposomes and microspheres [4,5].

In nasal drug delivery, the most important limitation factor is rapid mucociliary clearance, which is the cause of a limited contact period allowed for drug absorption through the nasal mucosa. Thus, mucoadhesive nano and micro-particles have been formulated to overcome the rapid mucociliary clearance, thereby increasing drug absorption through nasal cavity [6]. Chitosan is a natural polymer that has mucoadhesive properties because of its positive charges at neutral pH, which enable an ionic interaction with the negative charges of sialic acid residues on the mucus [7]. This highly mucoadhesive characteristics of chitosan provide a longer contact period for drug transport through nasal mucosa and prevents the clearance of the formulation via mucociliary clearance mechanism. Therefore, chitosan microspheres have been extensively evaluated as a drug delivery system. In this study, we aimed to formulate glibenclamide-loaded bioadhesive microspheres with chitosan and to investigate feasibility of glibenclamide nasal delivery with chitosan microspheres [8,9].

Chitosan microspheres have received considerable attention as nasal drug delivery systems. Chitosan, being biodegradable, biocompatible, and non-toxic and bioadhesive polymer is a suitable excipient for use in biomedical and pharmaceutical formulations [10,11]. Chitosan is a cationic polysaccharide, derived by the deacetylation of chitin. Chitosan is positively charged due to its amino group and able to interact strongly with the negatively charged mucus layer of the nasal epithelium. This is to provide a longer contact time for drug transport across the nasal membrane, before the formulation is cleared by the mucociliary clearance mechanism. In addition, chitosan has been shown to increase the paracellular transport of polar drugs by transiently opening the tight junctions between the epithelial cells [12,13].

Glibenclamide is a second-generation sulfonylurea used in the treatment of noninsulin-dependent diabetes. It is one of the most prescribed long-acting anti-hyperglycemic agents. Glibenclamide is classified as BCS class II drug, which means it has high permeability and poor water solubility. It has a short biological half life (3 to 5 h), with a log p value of 4.7 (o/w). Thus, it has poor aqueous solubility and undergoes oxidative hepatic first-pass metabolism to yield metabolites having no hypoglycemic activity [14-16].

In the present study chitosan microspheres intended for nasal delivery of glibenclamide were prepared by emulsification cross linking technique using glutaraldehyde (GLA) as the cross linking agent. Microspheres were characterized in terms of the particle size, morphological properties, production yield, drug content, encapsulation efficiency, and in vitro drug permeation.



Glibenclamide was received as a kind gift from Sun Pharmaceutical Industries Ltd. (Sikkim, India). Chitosan was provided by Sigma Aldrich, Steinheim, Japan. All other ingredients used were of analytical grade and were used without further purification. Spectrophotometric studies were carried out by using double-beam UV-spectrophotometer, Shimadzu, Pharma Spec 1700, Kyoto, Japan.


Preparation of mucoadhesive microspheres: Microspheres were prepared by the emulsification crosslinking technique. For the preparation of microspheres, 1%-3% w/v solutions of chitosan were prepared in acetic acid. Then drug and cross-linking agent were added in each chitosan solution to prepare a uniform dispersion. The drug dispersion was then extruded with the help of syringe and needle (24G) into liquid paraffin containing span 80 being kept under 100 rpm stirring. After 1 hr, the microspheres were separated by filtration and washed with petroleum ether to remove traces of paraffin oil. The microspheres were dried overnight in an oven at 40° C [7].

Experimental design: A 32 full factorial was applied to design the experiments. Concentration of chitosan and volume of glutaraldehyde was used as independent variables, whereas particle size, % mucoadhesion and % cumulative drug release were kept as dependent variables. Formulation F1- F10 was prepared using three different levels of chitosan and glutaraldehyde and response parameters were evaluated (Table 1).

Evaluation of glibenclamide loaded microspheres

Particle size: The optical microscope (HiconR, Grover Enterprises, Delhi) was used for measurement of particle size of microspheres and the mean particle size was calculated by measuring more than 100 particles with the help of a calibrated ocular micrometer (HiconR, Grover Enterprises, Delhi).

Determination of percentage yield: Percentage yield of glibeclamide containing microspheres was calculated by dividing actual weight of product after drying to total amount of glibenclamide and excipients used for the preparation of microspheres and are represented by following formula:

Percentage Yield =

\frac{Actual weight of microspheres after drying}{Total weight of the drug and excipients}\times 100

Determination of entrapment efficiency: 10 mg of microspheres were crushed and dissolved in required quantity of phosphate buffer pH6.4 and kept overnight. The samples were assayed for drug content by UV- spectrophotometer at 300 nm. The drug loading and entrapment efficiency was calculated using following formula:

Entrapment efficiency (%) =\frac{Mactual}{Mtheoretical}\times 100

Where M_{actual}is the actual glibenclamide content in weighed quantity of powder of microspheres and Mtheoretical is the theoretical amount of glibenclamide in microspheres calculated from the quantity added.

Swelling ability of microspheres: The swelling ability of microspheres was determined by allowing them to swell to their equilibrium in phosphate buffer of pH 6.4. Swelling was determined in triplicate by using the following formula:

\alpha =\frac{Ws-Wo}{Ws}

Where α is degree of swelling, Wo is initial weight of microspheres and Ws is the weight of microspheres after swelling.

In vitro mucoadhesion study: The mucoadhesive property was determined by falling liquid film technique. A freshly cut piece, 5 cm long, of goat nasal mucosa obtained from a local abattoir was cleaned by washing with isotonic saline solution. An accurate weight of microspheres was placed on mucosal surface, which was attached over a glass plate that fixed in an angle of 45° C relative to the horizontal plane and phosphate buffer of pH 6.4 warmed at 37° C was drawn at a rate of 5 mg/min over the tissue from the burette. One hour after administration of microspheres, the concentration of the drug in the collected perfusate was spectrophotometrically determined. The microspheres amount corresponding to the drug amount in perfusate was calculated. The adhered microspheres amount was estimated from the differences between the applied microspheres and the flowed microspheres amount. The ratio of the adhered microspheres was computed as percentage mucoadhesion [17].

In vitro drug permeation: The permeation of drug from microspheres was determined by using a Franz diffusion apparatus which consists of donor and receptor compartments. Goat nasal mucosa was used to keep the microspheres on the donor side, which allowed free diffusion of drug to the receptor compartment containing 10 ml phosphate buffer solution pH 6.4 (within the pH range in nasal cavity) and maintained at 37 ± 0.05° C. The receptor compartment was stirred with a magnetic stirring bar. At predetermined time intervals, aliquots (1 ml) were withdrawn from receptor cell and replaced with the same volume of fresh medium. The samples were diluted appropriately and assayed spectrophotometrically at 300 nm to analyze the drug permeation. Graph was plotted between percent cumulative drug permeation versus time. The release data generated was subjected to zero order, first order and Higuchi’s model(s) to understand the release kinetics of glibenclamide from microspheres. The model with highest correlation coefficient was considered to be the best fitted for release profile.

Selection of optimized formulation

The optimized formulation was selected on the basis of minimum particle size, maximum mucoadhesion and maximum cumulative drug permeation at 12 h, and was subjected to characterization studies.

Characterization of optimized formulation

Scanning electron microscopy: The surface morphology of the glibenclamide, placebo and glibenclamide loaded microspheres were examined by scanning electron microscopy. The samples were mounted directly onto the SEM sample holder using double sided sticking tape and images were recorded at the 100X magnification at the acceleration voltage of 10 kv.

Differential scanning calorimetry: Differential Scanning Calorimetry was performed on placebo microspheres, drug-loaded microspheres and Glibenclamide using differential scanning calorimeter instrument (NETZSCH DSC 200F3 240- 427-L, Japan). Samples were heated at the rate of 10° C min-1 over the temperature range of 30-300° C. An inert atmosphere was maintained by purging with nitrogen at the flow rate of 70 mL/ min.

Fourier transform infrared spectroscopy (ft-ir spectroscopy): Glibenclamide, Chitosan physical mixture and optimized formulation (F8) was examined using FTIR Spectrophotometer (Shimadzu FTIR-8400S Kyoto, Japan). The test sample diluted with KBr to get a final dilution of 1:10 was mounted into the instrument. The measurements were made in transmittance mode in the range of 400-4000 cm-1 against the background spectra of pure KBr by setting resolution of 4 cm-1 and 50 times accumulation.

Development of microspheres loaded in situ gel: Optimized formulation F8 was developed as micro particulate loaded in situ gel (thermoreversible) by cold method. In this method, specified amount of carbopol 934P was stirred in the calculated amount of distilled water at room temperature. The dispersion was cooled to 4° C by keeping it in a refrigerator. Pluronic F127 was added in different concentrations with continuous stirring at 50 rpm by Remi stirrer (thermostatically controlled magnetic stirrer, S.M. Scintific Instruments (P), Ltd, India). Dispersion was then stored in a refrigerator overnight to clear solution. The microspheres containing 50 mg equivalent Glibenclamide were incorporated into the in situ gel. Thereafter, gel formulation was stored in a refrigerator so that it remains in sol form.

Evaluation of glibenclamide loaded microspheres incorporated in in situ gel

Clarity: The clarity of all formulations was determined by visual inspector under black and white background.

Gelling temperature: It is defined as the temperature at which liquid phase makes the transition to a gel. 2ml of sol were transfer to a test tube, immersed in water bath. The temperature of water bath was increased in increment of 1° C and left to equilibrate for five minute at each new setting. The sample was examined for gelation which was said to have occurred when the meniscus would have no longer move upon tilting test tube through 90° C.

pH and viscosity: One ml quantity of gel formulation was transferred to the 10ml volumetric flask and diluted by using distilled water to make volume up to 10ml. the pH of resulting solution was determined by using digital pH meter, model 111 E(HICON, New Delhi, India). The viscosity of in situ gel was measured using Brookfield viscometer DV-II + Pro coupled with S-94 spindle at 100 rpm at 4 ± 1° C.

Drug content: One ml of gel was extracted with 10 ml phosphate buffer pH 6.4 by vortexing (vortex shaker, HICON, India). Further dilution were made with phosphate buffer pH 6.4 and analyzed by UV spectrophotometer at 300 nm.

Gel strength: Gel strength was measured by placing 50 g of formulation in a 100ml graduated cylinder and gelled at 37° C using thermostat. A weight of 35 g was placed on to the gelled solution and allowed to penetrate 5 cm in the gel. Time taken by weight to sink 5 cm was measured.

Ex-vivo permeation: The in vitro permeation study was performed using goat nasal mucosa. The nasal mucosa was collected from local slaughter house and washed three times with phosphate buffer pH 6.4. The extraneous tissues were removed with surgical blade to get the mucosa. The prepared nasal mucosa was inserted in Franz diffusion cell having permeation area of 3.14 cm2 . Stabilization was performed by stirring the receiving medium with magnetic stirrer for 30 minute in phosphate buffer pH 6.4. After stabilization the phosphate buffer was replaced with fresh media i.e. 18 ml of phosphate buffer pH 6.4 at 34° C. Then the formulation equivalent to 5mg of glibenclamide was replaced in the donor chamber. At predetermined time points i.e. at 0,1……..8h, 1ml sample were withdrawn from the acceptor compartment and replaced with an equal volume of the phosphate buffer pH 6.4. the amount of permeated drug was determined by using UV spectrophotometer at 300 nm.


Histological studies were conducted to determine the effect of formulation on nasal mucosa. Goat nasal mucosa was collect from the local slaughter house and transported to laboratory in normal saline at cold condition within 1h of slaughtering. The nasal mucosa was incubated in optimized G5, phosphate buffer saline pH6.4 as negative and 75% isopropyl alcohol as positive control for 30minute. The mucosa washed with phosphate buffer pH6.4 then immediately fixed with 10% v/v formalin solution for 24h, their after embedded in paraffin. Paraffin sections (7 µm) were cut on glass slides and stained with hematoxylin and eosin. Then to detect any damage to the tissue, sections were observed through photomicrograph (HICON enterprises, New Delhi, India) at 100X magnification.


Characterization of glibenclamide loaded microspheres

Glibenclamide loaded cross linked chitosan microspheres were prepared with spray-drying method. The characteristics of the microspheres are presented in Table (3).

Particle Size: The largest particle size was found in formulation F7 (75.66 µm) (Table 3). F3 showed least particle size (38.66 µm) followed by F2 (40.33 µm). A particle size more than 10 µm and less than 100 µm is desirable for nasal deposition thus all the formulations were suitable for nasal delivery. Formulation F7 exhibited larger particle size (75.66 µm) than F4 (52.66 µm) because of higher concentration of polymer. Analysis of the effect of independent variables on the particle size revealed that on increasing the concentration of the polymer (chitosan) the particle size increased, as high concentration of chitosan increases the viscosity of polymeric dispersion and larger droplets are formed. As the concentration of cross linking agent was increased, the particle size decreased due to formation of rigid network structure.

Percentage yield: Percentage yield of microspheres ranged within 68.66 to 84.57% (Table 3) indicating efficiency of the process. Considering the design as the concentration of polymer increased, % yield increased.

In vitro mucoadhesion: The mucoadhesion of glibenclamide loaded nasal microspheres closely varied between 67.9 ± 0.675 to 87.20 ± 1.101 (Table 3) and was dependent on polymer concentration. The percent mucoadhesion was found to decrease from 87.2 ± 1.101 to 67.9 ± 0.675 with gradual decrease in concentration of chitosan from 3 to 1% w/v. F3 showed lowest mucoadhesion (64.3 ± 0.650) containing more concentration of cross linking agent but F8 exhibited highest mucoadhesion (87.2 ± 1.101) followed by F3 (64.3 ± 0.650). As the concentration of chitosan increased the mucoadhesion ability of the microspheres increases due to enhanced binding of chitosan molecules with Mucin. As a result the formulation will be able to resist physiological process of mucociliary clearance in vivo. The mucoadhesive property of microspheres decreased on increasing the concentration of cross-linking agent. Pre-requisite for a good mucoadhesion is the high flexibility of polymer backbone structure and its polar functional groups. Such flexibility of the polymer chain is reduced if the polymer molecules are cross-linked either with each other or with cross-linking agent. The decrease in flexibility imposed upon polymer chain by cross-linking makes it more difficult for cross-linked polymer to penetrate the mucin network. Thus cross-linking effectively limits the polymer chain that can penetrate the mucus layer and could possibly decrease mucoadhesion strength.

In vitro drug permeation study: Permeation study of glibenclamide loaded nasal microsphere of all formulation was performed in the pH of nasal cavity (phosphate buffer pH 6.4) for 12 hrs using goat nasal mucosa in Franz diffusion cell [18]. It was found that, at the end of 12hrs formulation F5 and F8 showed highest drug permeation in phosphate buffer pH 6.4 i.e 77.94 ± 2.869 and 80.81 ± 2.205 respectively (Figure 1). This shows that when particle size increased, drug permeation was decreased. In large microspheres permeation was decreased due to increase in diffusional path length which the drug molecules have to travels (Sultana et al., 2010). But F8 shows high drug permeation in comparison of F5 this may be due to less drug entrap in microspheres of F5 formulation because of less particle size. The results showed that optimized formulation showed highest and prolonged drug release as compared to other formulation and in optimized formulation drug release rate was controlled by the presence of polymer (chitosan). Target flux of microsphere formulation was obtained 17.579 µg/cm²/hr which was achieved in optimized F8 formulation.

Data analysis: On the basis of the data obtained from the optimized formulations, a general statistical model can be depicted with respect to the above data. The model developed can be characterized by using the polynomial equation representing the respective response data. This can be given as follows:

Particle size = + 49.67 + 13.83X1 - 4.8X2 - 2.255X1X2 + 4.83X12 - 0.17X2^{2}

% Mucoadhesion = + 74.67 + 8.80X1 - 1.53X2

% Cumulative drug permeation = + 78.29 + 6.78X1 + 4.59X2 + 0.34X1X2 - 4.39X12 - 9.77X2^{2}

After removing the non-significant values the equation found out is known as transformed equation or final equation. These are as follows:

Particle size = + 49.78 + 13.83X1 - 4.8X2 - 2.255X1X2 + 4.83X1^{2}

% Mucoadhesion = + 74.67 + 8.80X1

% Cumulative drug permeation = + 78.29 + 6.78X1 + 4.59X2 - 4.39X12 - 9.77X2^{2}

From the above polynomial equations, 3D response surface graphs of the respective responses were generated, which were used to predict the responses of dependent variables at the intermediate levels of independent variables.

Response-surface analysis: Three dimensional response surface plots drawn for the graphical optimization of glibenclamide loaded nasal system are presented in Figure (2). Figure (2a) shows that when concentration of chitosan (X1) and volume of glutaraldehyde (X2) increases simultaneously than particle size was increases. Same effect was observed in Figure (2b) when concentration of chitosan (X1) and volume of glutaraldehyde (X2) increases simultaneously than Mucoadhesion was also increases. In case of % CDP, when concentration of chitosan increases and volume of glutaraldehyde decreases, then % CDP increases.

Validation of design: For the validation of 32 full factorial design one extra check point was formulated and evaluated on the basis of their particle size, % mucoadhesion, and % cumulative drug release. The results were found that the experimental values were close to the predicted values. (Table 4) lists the composition of the extra check points, the predicted and experimental values of the entire response variable and the percentage error, which is calculated by the following formula;

Percentage error =\frac{Predicted value-Actual value}{Predicted value}\times 100

Upon comparison of the predicted values to the experimental values, the predicted error was found. Thus the low magnitude of error in the current study indicates a high prognostic ability of nasal microspheres of glibenclamide and also proved that 32 full factorial design was validated.

Selection of optimized formulation

The formulation F8 was optimized on the basis of minimum particle size, maximum mucoadhesion and % cumulative drug permeation which were found to be 69.11 µm, 87.20 ± 1.101 % and 80.81 ± 2.205 % respectively. The in-vitro drug permeation data was fitted to different kinetics models namely zero order, First model, Higuchi model, Hixon-Crowell model and Peppas model. Zero order model was considered as the best fitted model with the highest value of coefficient of determination which is as r2 = 0.9729 in pH 6.4 (Table 3), which shows that the drug was released in controlled manner in the simulated nasal pH.

Characterization of optimized formulation

Scanning electron microscopy: The placebo and drug loaded microspheres were nearly spherical in appearance (Figure 3). Placebo and glibenclamide microspheres were found to be discrete and with rough surface. SEM micrograph reveals no difference between the surface of placebo and drug loaded microspheres, explaining the dispersion of drug in the polymeric matrix.

Differential scanning calorimetry (DSC): DSC thermogram of the glibenclamide showed sharp peak at 172° C due to its melting, which is consistent with literature report (B.P 2004). Placebo microspheres showed an exothermic peak at 92° C. This indicated that the exotherm at 92° C corresponded to thermal decomposition of the chitosan. In glibenclamide loaded microspheres characteristic peak of glibenclamide was not observed at 172° C, suggesting that glibenclamide was molecularly dispersed in matrix (Figure 4).

Fourier transforms infrared spectroscopy (FTIR): The FTIR studies were carried out to assess any possible interaction between drug and polymer. The FTIR spectrum of glibenclamide was done by using KBR pellet technique. Glibenclamide and chitosan showed characteristic peak at range of 400-4000 cm1 . Glibenclamide showed prominent peaks at wave numbers were 3315 cm-1 due to stretching of N-H group. Sharp peak was observed at 2910 cm-1 indicated C-H stretching vibration. The peak at 2350 cm-1 and 1714 cm-1 showed stretching due to O-H and C=O group. The dominant IR peak of chitosan was observed at 2920 cm-1 due to presence of C-H group, peak at 1310 cm-1 due to N-acetyl glucosamide and -CO group stretching at 1094 cm-1. The spectral analysis of optimized formulation (F8) as shown in Figure (5) - showed characteristic bands of glibenclamide at 3315 cm-1, 2931 cm-1, 1714 cm-1 corresponding to -NH stretching, C-H stretching and C=O stretching respectively (Pavia et al., 2007). These characteristic stretching vibrations were also present in physical mixture (Figure 5) without any significant shift in the wave number of peaks and changes in the intensity of the peaks indicating no interaction between drug and polymer.

Formulation of glibenclmide loaded microspheres incorporating in in situ gel

The optimized formulation (F8) was further incorporated into thermoreversible in situ gelling system with the aim to achieve more controlled release of microspheres from gelling system. The evaluation characteristics of different formulations of glibenclamide loaded microspheres incorporated in in situ gel (G1-G5) prepared using cold method (Table 2).

Evaluation of glibenclamide loaded microspheres incorporated in in situ gel

Clarity: All the formulations were found to be clear. There were no residue matters present which can harm nasal mucosa and affect the syringeability of the formulations [19].

Gelling temperature: The gelling temperature of the formulations was ranged from 27.2-35.9° C (Table 6). A gelling temperature is suitable in the range of 25° C to 37° C. The gel having gelling temperature lower than 25° C, leads to difficulty in manufacturing, handling and administering and gel having gelling temperature higher than 37° C, it would not form a gel at the temperature of the nasal cavity. The results revealed that the gelling temperature decreased with increase in the concentration of thermo sensitive polymer Poloxamer 407 due to formation of larger number of the micelles that occupy larger volume at lower temperature [20]. The mucoadhesive agent carbopol 934 P also causes lowering of gelling temperature because of its ability to bind to poly (ethylene) oxide chains present in Poloxamer 407 molecule, thus promoting dehydration and cause an increase in entanglement of adjacent molecule with more extensive intermolecular H-bonding [21]. Below the transition temperature Poloxamer 407 solutions allow a precise and comfortable delivery in the nasal cavity where thermo gelation occurs. Immediate gelling increases residence time.

pH and viscosity: pH is a very important factor for nasal formulations. The normal physiological pH of the nasal mucosa ranges from 4.5-6.5. But the nasal mucosa has capability to tolerate pH between 3 to 10. The pH of all the formulations was ranged from 5.2 ± 0.20-5.8 ± 0.26 i.e, which is within the range of nasal mucosa and can be easily tolerated and thus causes no irritation to it. As alkaline pH inactivates the lysozyme secreted by nasal cells, hence makes the nasal tissue susceptible to microbial infection and lower pH acts as hypertonic solution, which causes the shrinkage of epithelial cells and also inhibits ciliary activity. Viscosity of all the formulations lies between 45.6 ± 0.15-67.7 ± 0.15 cps. The formulations should have an optimum viscosity, which will allow its easy administration into nasal cavity, as a liquid, which will then undergo rapid sol to gel conversion and it helps in increasing the nasal residence time of the formulation by decreasing the mucociliary clearance [21].

Drug content: Drug content of all formulations was found to be in the range of 81.24-93.91% (Table 6). Formulation G5 was found to contain high drug content (93.91 ± 2.07%) when compared to other in situ gel formulations.

Gel strength: All the formulations showed a gel strength value in range 27.36-48.40 (Dyne/cm2 ) (Table 6) which was acceptable for nasal delivery as the gel strength values between 25 to 50 sec. were considered sufficient. The gel strength was found to be affected by concentration of Poloxamer 407. It has been reported that the gel strength duration of less than 25 sec is not be able to retain its integrity and can erode rapidly while gels with strength greater than 50 sec are too stiff which may cause discomfort to the mucosal surfaces or may damage it [17].

In vitro drug permeation study: In vitro drug permeation studies of glibenclamide from optimized formulation F8, G5 and control gel were studied using goat mucosa via Franz diffusion apparatus (Figure 6) [22]. The percentage cumulative drug permeation from in situ gel formulation (G5) at 12 h was found to be 69.27 ± 0.196. The permeation profile of G5 was compared to optimized microspheres formulation (F8) and control gel of same concentration of Poloxamer 407 (18% w/v) as compared to G5. A significant difference was observed between the permeation profiles. The permeation profile was obtained indicate the greater sustained action of developed microparticulate in situ gel G5 as compared to the control gel and microparticulate formulation (F8) because of presence of Poloxamer 407 in the gel which retards the drug release rate and thus permeation slightly decreases due to reduction in dimension of water channels [23].

This study also confirms the importance of chelating calcium for enhancing the permeation of glibenclamide across the mucosa. Anionic polymer carbopol is reported to demonstrate permeation - enhancing properties as it is able to bind Ca2+ of the nasal mucosa [24-28]. When carbopol concentration is increased, it results in increased concentration of ionized carboxyl group which causes conformational changes in the polymeric chain. Decoiling of the polymer chain occur due to electrostatic repulsion of the ionized carboxyl group, which results in relaxation of polymer network. During this stage, drug is rapidly dissolved and diffused from the gels due to very high swelling of the ionized carbopol [21].

Table 1: 3^{2}Full Factorial Design Layout for Preparation of Glibenclamide loaded microspheres.

Formulation code Glibenclamide (mg) Chitosan (%w/v) (X1) GLA (ml) (X2) Dependent variables
F1 100 1(-) 1(-) Particle size (Y1)
% Mucoadhesion (Y2)
% CDP (Y3)
F2 100 1(-) 2(0)
F3 100 1(-) 3(+)
F4 100 2(0) 1(-)
F5 100 2(0) 2(0)
F6 100 2(0) 3(+)
F7 100 3(+) 1(-)
F8 100 3(+) 2(0)
F9 100 3(+) 3(+)
F10* 100 1.5 1.5
*extra design check point formulation

Table 2: Formulation composition of Glibenclamide loaded microparticulate in situ gel.

Formulation code Concentration of Poloxamer 407 (%w/v) Concentration of carbopol 934P (%w//v)
G1 14 0.5
G2 16 0.5
G3 18 0.5
G4 20 0.5
G5 22 0.5

Table 3: Characterization of mucoadhesive glibenclamide loaded microspheres.

Code Mean Particle size (µm) P. Yield (%) Drug Entrapment (%) Entrapment Efficiency (%) Mucoadhesion (%±SD) Swelling Index (%±SD) Cumulative drug permeation (%±SD)
F1 44 68.66 68.11±0.300 33.06±0.145 67.9±0.675 0.210±0.0095 57.16±0.381
F2 40.33 79.81 69.54±1.761 17.42±0.443 65.9±1.305 0.182±0.0071 67.33±0.303
F3 38.66 82.85 78.94±2.016 13.61±0.348 64.3±0.650 0.169±0.0056 61.49±0.172
F4 52.66 84.57 78.12±2.019 13.02±0.104 75.1±1.014 0.293±0.0081 64.10±0.502
F5 50.66 81.28 80.27±0.282 14.10±0.017 74.3±1.069 0.273±0.0023 77.94±2.869
F6 45 81.40 82.52±1.184 20.27±0.290 73.6±1.365 0.238±0.0037 73.27±0.479
F7 76 79.00 82.17±0.834 20.80±0.210 83.9±1.708 0.366±0.0036 65.90±0.172
F8 68 73.64 85.27±0.550 38.57±0.250 87.2±1.101 0.340±0.0050 80.81±2.205
F9 61 77.66 82.06±0.960 35.21±0.406 79.8±0.556 0.320±0.0051 75.76±0.335
F10* 54 79.78 78.63±0.571 20.74±0.149 72.7±1.20 0.250±0.0058 80.52±0.334

Table 4: Comparison of experimental results with predicted responses of glibenclamide loaded nasal microspheres formulations.

F. code Composition Response Predicted Value Experimental Value Error (%)
X1 X2
F 10* 1.5 1.5 Particle size (µm) 54.81 54 1.47
% Mucoadhesion 78.30 78.28 0.025
% Cumulative Drug permeation 80.52 80.32 0.248

Table 5: Model fitting on the release data of glibenclamide from optimized formulation (F8).

Simulated pH Result Zero Order First Order Higuchi Peppas Hixon-Crowel
6.4 r2 0.9729 0.9715 0.8812 0.9368 0.9521
B 7.55 0.063 2.730 1.647 0.179
A 1.78 2.068 1.865 0.324 4.781

Table 6: Evaluation parameters for glibenclamide loaded mucoadhesive microspheres incorporated in in situ gel.

Formulation code Clarity pH Gelling temp. (°C) Viscosity of sol (cps) Drug content (%) Gel streanth (dyne/ cm2)
G1 Clear 5.6±0.25 35.9±0.55 45.6±0.15 85.57±2.50 27.36±0.55
G2 Clear 5.8±0.26 32.2±0.35 48.6±0.05 81.24±3.19 36.23±0.45
G3 Clear 5.5±0.26 30.5±0.80 55.6±0.20 89.07±1.50 41.23±0.30
G4 Clear 5.4±0.20 29.3±0.95 61.3±0.05 84.57±2.67 44.90±0.36
G5 Clear 5.2±0.20 27.2±0.45 67.7±0.15 93.91±2.07 48.40±0.45



The integrity of optimized microparticulate in situ gel formulation (G5) was evaluated using histological sections of sheep nasal mucosa. After incubating nasal mucosa in 75 % isopropyl alcohol, an irritant damage of the epithelium from the nasal mucosa was observed. But no significant effect on the microscopic structure of mucosa was seen when microscopic observation was done for the optimized micro particulate in situ gel formulation (G5). As shown in Figure (7) neither cell necrosis nor removal of the epithelium from the nasal mucosa was observed after permeation of gel. There were no alterations in epithelium layer, basal membrane and superficial part of sub mucosal blood vessels. Thus it can be said that the in situ gel formulation can be safely administered via nasal route.


Developed glibenclamide loaded mucoadhesive microspheres incorporated in in-situ gel (G5) can be considered as an effective and superior doses form. It may overcome the drawbacks of commercial formulations. The developed formulation show longer duration of action (up to 12h). Furthermore, presence of poloxamer 407 as an in situ gelling polymer had sufficient mucoadhesive properties that result in increase residence time of drug with the nasal mucosa due to transformation of microspheres into gel which is not easily clear by mucociliary clearance [29-34].


1. Djupesland PG. Nasal drug delivery devices: characteristics and performance in a clinical perspective-a review. Drug Deliv Transl Res. 2013; 3: 42-62.

2. Patil SB, Sawant KK. Chitosan microspheres as a delivery system for nasal insufflations. Colloids Surf B Biointerfaces. 2011; 84: 384-389.

3. Mygind N, Dahl R. Anatomy, physiology and function of the nasal cavities in health and disease. Adv Drug Deliv Rev. 1999; 29: 3-12.

4. Ozsoy Y. Particulate carriers for nasal administration. In: Kumar MNVR, ed. Handbook of particulate drug delivery. CA: American Scientific Publisher. 2008; 143-64.

5. Chowdary KP, Rao YS. Mucoadhesive microspheres for controlled drug delivery. Biol Pharm Bull. 2004; 27: 1717-1724.

6. Gavini E, Rassu G, Sanna V, Cossu M, Giunchedi P. Mucoadhesive microspheres for nasal administration of an antiemetic drug, metoclopramide: In-vitro/ex-vivo studies. J Pharm Pharmacol. 2005; 57: 287-294.

7. Gavini E, Hegge AB, Rassu G, Sanna V, Testa C, Pirisino G, et al. Nasal administration of carbamazepine using chitosan microspheres: In vitro/in vivo studies. Int J Pharm. 2006; 307: 9-15.

8. Singla AK, Chawla M. Chitosan: Some pharmaceutical and biological aspects an update. J Pharm Pharmacol. 2001; 53: 1047-1067.

9. Soane RJ, Frier M, Perkins AC, Jones NS, Davis SS, Illum L. Evaluation of the clearance characteristics of bioadhesive systems in humans. Int J Pharm. 1999; 178: 55-65.

10. Artursson P, Lindmark T, Davis SS, Illum L. Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm Res. 1994; 11:1358-1361.

11. Sinha VR, Singla AK, Wadhawan S, Kaushik R, Kumria R, Bansal K, et al. Chitosan microspheres as a potential carrier for drugs. Int J Pharm. 2004; 274: 1-33.

12. Hafner A, Filipovic-Grcic J, Voinovich D, Jalsenjak I. Development and in vitro characterization of chitosan-based microspheres for nasal delivery of promethazine. Drug Dev Ind Pharm. 2007; 33: 427-436.

13. Asane GS, Nirmal SA, Rasal KB, Naik AA, Mahadik MS, Rao YM. Polymers for mucoadhesive drug delivery system: A current status. Drug Dev Ind Pharm. 2008; 34: 1246-1266.

14. Groop L, Wahlin BE, Totterman KJ, Melander A, Tolppanen EM, Fyhrqvist F, et al. Pharmacokinetics and metabolic effects of glibenclamide and glipizide in type 2 diabetes. Eur J Clin Pharmacol 1985; 28: 697-704.

15. Wei H, Lobenberg R. Bio relevant dissolution media as a predictive tool for glyburide: a class II drug. Eur J Pharm Sci 2006; 19: 45-52.

16. Philip AK, Srivastava M, Pathak K. Buccoadhesive gels of glibenclamide: a means for achieving enhanced bioavailability. Drug Deliv. 2009? 16: 405-415.

17. Arora P, Sharma S, Garg S. Permeability issues in nasal drug delivery. Drug Discov Today. 2002; 7: 967-975.

18. Boltan S. Pharmaceutical statistics; Practice and clinical applications. Marcel Dekker. New York. 1997; 215-256.

19. Goodman, Gilman. The pharmacological basis of therapeutics. Mc graw Hill Medical Publishing division, 11th edition, New York. 2006: 687-712.

20. Huh Y, Cho HJ, Yoon IS, Choi MK, Kim JS, Oh E, et al. Preparation and evaluation of spray-dried hyaluronic acid microspheres for intranasal delivery of fexofenadine hydrochloride. Eur J Pharm Sci. 2010; 40: 9-15.

21. Illuma L. Nasal drug delivery-possibilities, problems and solutions. J Control Release. 2003; 87: 187-198.

22. Illum L. Mathiowitz E. Chickering DE, Lehr CM. Bioadhesive formulations for nasal peptide delivery: Fundamentals, Novel Approaches and Development. Marcel Dekker. New York. 1999: 507-539.

23. Kumar A, Bali V, Kumar M, Pathak K. “Comparative evaluation of porous versus non porous mucoadhesive films as buccal delivery system of glibenclamide”. AAPS Pharm SciTech. 2013; 14: 1321-1332.

24. Pathak K. Mucoadhesion; A prerequisite or a constraint in nasal drug delivery. Int J Pharm Investig. 2011; 1: 62-63.

25. Patil SB, Sawant KK. Development, Optimization and in vitro evaluation of alginate mucoadhesive microspheres of carvedilol for nasal delivery. J Microencapsul. 2009; 26: 432-443.

26. Pavia DL, Lampman GM, Kriz GS, Vyvyan IR, Spectroscopy. 7th edition. 2007; 38: 37.

27. Rhees RW, Graaff KM. “Schaum’s Outlines of theory and problems of Human Anatomy and Physiology”. Edition 2nd, Tata McGraw-Hill publishing company Limited, New Delhi. 1997; 329.

28. Remington. “The Science and Practice of Pharmacy”. Edition 21st, Vol. II, Lippincott Williams and Wilkins. 2007; 1454.

29. Tripathi KD. ‘Essential of medical pharmacology’ Edition 5th, Jaypee Brothers, medical publishers (p) Ltd., New Delhi. 2003; 132-135.

30. Ugwoke MI, Agu RU, Verbeke N, Kinget R. Mucoadhesive Drug Delivery: Background, applications, trends and future perspectives. Adv Drug Deliv Rev. 2005; 57: 1640-1665.

31. Vyas SP, Khar RK. “Controlled drug delivery concepts and advances”. 1st ed., New Delhi, Vallabh Prakashan. 2002; 417-55.

32. The Indian Pharmacopoeia 1996, Govt. of India, Ministry of Health and Family Welfare, Controller of Publications, New Delhi. Vol. 1, pp. 347.

33. Wu S. Formulation of adhesive bond, polymer interface and adhesion. Marcel Dekker Inc, New York, 1982; 359-447.

34. Zhou M, Donovan MD. “Intranasal mucociliary clearance of putative bioadhesive polymer gels”. Int J Pharm. 1996; 135: 115-125.

Received : 30 Nov 2016
Accepted : 25 Jan 2016
Published : 26 Jan 2017
Annals of Otolaryngology and Rhinology
ISSN : 2379-948X
Launched : 2014
JSM Schizophrenia
Launched : 2016
Journal of Nausea
Launched : 2020
JSM Internal Medicine
Launched : 2016
JSM Hepatitis
Launched : 2016
JSM Oro Facial Surgeries
ISSN : 2578-3211
Launched : 2016
Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
TEST Journal of Dentistry
ISSN : 1234-5678
Launched : 2014
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
Author Information X