Preparation and evaluation of mucoadhesive cefdinir microcapsules.

The mucoadhesive microcapsules were prepared by using various concentrations of three different mucoadhesive polymers, namely, chitosan, Carbopol 934P, and methyl cellulose as wall materials and cefdinir as the core material employing orificeionic gelation method. The prepared microcapsules were characterized by scanning electron microscope (SEM) and Fourier transform infrared spectrometry (FT-IR). The prepared microcapsules were found to be spherical with particle size ranging from 765±20 to 985±10 μm and encapsulation efficiencies in the range of 55%-92%. The formulation containing Carbopol 934P as mucoadhesive polymer was found to be best with particle size 946±10 μm. The ex vivo wash-off test showed that the mucoadhesion after 1 h was 80% and the in vitro drug release was extended for more than 12 h. FT-IR spectra indicate that there was no interaction between drug and the polymers used in the formulation. Cefdinir is better absorbed from the upper part of the gastrointestinal tract, it suffers from low oral bioavailability (20-30%), shorter biological half-life (1-2 h), and less transit time. Thus, it can be concluded that microcapsules prepared using Carbopol 934P have promising properties for use as mucoadhesive carrier to increase the residence time of cefdinir.

INTRODUCTION

The technique of mucoadhesion utilizes the property of bioadhesion of certain polymers that become adhesive on hydration and hence mucoadhesive drug delivery systems can be used to target the drug to a particular region of the body for controlled release of drug for extended periods of time.[12] Most of the hydrophilic polymers attract water from the mucous gel layer adherent to the epithelial surface thus adhering the mucosal surface. This is the simple mechanism of adhesion and is called as “adhesion by hydration.” Various types of forces, such as hydrogen bonding exist between the adherent polymer and the substrate, that is mucus, at the molecular level.[3-8] Bioadhesion is an interfacial phenomenon, which involves two materials, one of which must be biological, and these are held together by interfacial forces.[9] Carbopol polymers are known to create a strong bond with the mucous membrane, which results in strong bioadhesion.[10-13]

Chitin is the second highly abundant natural biopolymer, which is derived from the exoskeletons of crustaceans and also from cell walls of fungi and insects.[14] Chitosan is a product derived from N-deacetylation of chitin in the presence of hot alkali. It is widely used due to its versatile biological activity, excellent biocompatibility, and complete biodegradability with low toxicity.[15] The mucoadhesive property of chitosan is due to ionic interactions between the positively charged primary amino groups on the polymer and negatively charged sialic acid and sulfonic acid substructures of the mucus.[16]

Cefdinir is an expanded-spectrum, oral, third-generation cephem antimicrobial agent active against Gram-positive and Gram-negative bacteria.[1718] It is used in the treatment of acute chronic bronchitis, rhinosinusitis, and pharyngitis and uncomplicated skin and skin-structure infections in adults and adolescents; it is indicated for acute otitis media, acute sinusitis, and community-acquired pneumonia.[1920] Cefdinir requires controlled release because of its short biological half-life of ~1.5 h.[21] In this study, cefdinir mucoadhesive microcapsules were prepared using various concentrations of three different mucoadhesive polymers employing orifice-ionic gelation method.

MATERIALS AND METHODS

Materials

Cefdinir was a gift sample from (Aurobindo Pharmaceuticals Limited, Hyderabad, India). Carbopol 934P was a gift sample from (Noveon, Inc., USA). Chitosan of molecular weight 150–650 kDa and deacetylation degree of 80% was a gift sample from (Degussa, Germany). Sodium alginate of viscosity 4000 cps (Qualikems Fine Chemicals Pvt. Ltd, New Delhi, India), methyl cellulose of viscosity 450 cps (Qualikems Fine Chemicals Pvt. Ltd, New Delhi, India), and calcium chloride (Universal Laboratories Pvt. Ltd, Mumbai, India) were obtained from commercial sources. All other reagents used were of analytical grade.

Preparation of microcapsules

Cefdinir mucoadhesive microcapsules were prepared employing sodium alginate in combination with three mucoadhesive polymers—chitosan, Carbopol 934P, and methyl cellulose—as coat materials. The polymer concentrations used were in the range of 50–350 mg. An orifice–ionic gelation process, which has been extensively used to prepare large alginate beads, was employed to prepare the microcapsules.[2223]

Sodium alginate (200 mg) and the mucoadhesive polymer were dissolved in 10 mL of purified water and mixed thoroughly to form homogeneous polymer solution. The active substance, cefdinir (100 mg), was added to polymer solution and mixed thoroughly with a stirrer to form a viscous dispersion. The resulting dispersion was then added dropwise manually into calcium chloride (2% wt/vol) solution using a syringe with a needle of size no. 18. The added droplets were retained in the calcium chloride solution for 15 min to complete the curing reaction and to produce spherical rigid microcapsules. The microcapsules were collected by decantation, and the product thus separated was washed repeatedly with water and dried at 45°C for 12 h. The microcapsules prepared along with their coat composition are listed in Table 1.

Table 1

Formulations of microcapsules with different coat compositions

Estimation of cefdinir

Cefdinir content in the microcapsules was estimated by a UV spectrophotometric method (Systronics, 2202, Ahmedabad, India) based on the measurement of absorbance at 277 nm in phosphate buffer of pH 6.8. The method was validated for linearity, accuracy, and precision.

Microencapsulation efficiency

Microencapsulation efficiency was calculated using the following formula.[24]

Scanning electron microscopy

The microcapsules were observed under a SEM (FEI-Philips XL-30). They were mounted directly onto the SEM sample stub using double-sided sticking tape and coated with gold film of thickness 200 nm under reduced pressure of 0.001 mmHg.

In vitro drug release study

Release of cefdinir from the microcapsules was studied in 900 mL of 0.1 N HCl for first 2 h and in 900 mL of phosphate buffer pH 6.8 for the next 10 h using a United States Pharmacopeia (USP) apparatus I Basket type (Electrolab, TDT-08L, India) at 100 rpm and 37°C ± 0.5°C. A sample of microcapsules equivalent to 50 mg of cefdinir was used in each test. Samples of dissolution fluid were withdrawn through a filter of 0.45 μm at different time intervals and were assayed at 277 nm for cefdinir content using a UV spectrophotometer. The drug release experiments were conducted in triplicate.

Mucoadhesion testing by ex vivo wash-off test

The mucoadhesive property of the microcapsules was evaluated by an ex vivo adhesion testing method known as the wash-off method.[5] Freshly excised pieces of intestinal mucosa of 2 cm2 from pig were mounted onto glass slides of 3 × 1 inch with cyanoacrylate glue. Two glass slides were connected with a suitable support. About 50 microcapsules were spread onto each wet rinsed tissue specimen, and immediately thereafter the support was hung onto the arm of a USP tablet disintegrating test apparatus (Electrolab, Mumbai, India).When the disintegrating test machine was operated, the tissue specimen was given a slow, regular up-and-down movement in the test fluid at 37°C contained in a 1 L vessel of the machine. At the end of 30 min, at the end of 1 h, and at hourly intervals up to 12 h, the machine was stopped and the number of microcapsules still adhering to the tissue was counted. The test was performed at both gastric pH 0.1 N HCl, pH 1.2 and intestinal pH phosphate buffer, pH 6.8.

Fourier transform infrared spectroscopy

Infrared spectra were recorded between 4000 and 450 cm-1 by Perkin–Elmer FT-IR spectrometer SPECTRUM 1000 (Norwalk, USA). Each sample is mixed with Potassium bromide (KBr) in the ratio of 1:100 and compressed at 70 kN in a Perkin–Elmer hydraulic press for the preparation of pellets. These pellets are then analyzed in the Perkin–Elmer FT-IR spectrometer (SPECTRUM 1000, Norwalk, USA).

RESULTS

Cefdinir microcapsules with a coat consisting of sodium alginate and a mucoadhesive polymer, such as chitosan, methylcellulose, or Carbopol 934P, in different ratios could be prepared by the orifice–ionic gelation process. Microcapsules could not be prepared with mucoadhesive polymer alone because of their hydrophilic nature. The microcapsules were evaluated for drug content, particle size, encapsulation efficiency, in vitro drug release, mucoadhesion testing by ex vivo wash-off test, and interaction between drug and excipients by FT-IR. Formulations were prepared with different coat compositions were given in Table 1. The particle size, drug content, and encapsulation efficiencies of all the formulations were given in Table 2.

Table 2

Particle size, percentage drug content, and microencapsulation efficiency of formulations F1–F12

Ex vivo Wash-off Test

The best formulation from each polymer was selected and their mucoadhesion was tested by ex vivo wash-off test. The ability of the microcapsules to adhere the intestinal mucosa of pig was compared with the microcapsules prepared with nonmucoadhesive material, that is, ethyl vinyl acetate. The test was performed for 12 h in both 0.1 N HCl and pH 6.8 phosphate buffer using USP tablet disintegrating test machine. The results are shown in Table 3.

Table 3

Results of ex vivo wash-off test to assess mucoadhesive properties of the microcapsules prepared

Morphology of Carbopol 934P Microcapsules

The surface morphology of cefdinir microcapsules prepared by Carbopol 934P was seen by SEM. The surface morphology was done for formulation 8 with a magnification of ×60 [Figure 1]. The microcapsules were spherical-shaped and the surface of microcapsules was smooth.

Figure 1

Scanning electron micrographs of Carbopol 934P microcapsules (F8)

In vitro Drug Release Studies

Cefdinir release from the prepared microcapsules was studied in 0.1 N HCl for 2 h and in pH 6.8 phosphate buffer for 10 h. The drug release from the microcapsules was found to be slow and based on the composition of the coat, the drug release followed zero-order kinetics (R2 > 0.90). Microcapsules with coat containing alginate and chitosan gave relatively fast release when compared with others. The order of increasing release rate observed with various microcapsules was alginate–Carbopol 934P < alginate–methylcellulose < alginate–chitosan. The drug release from microcapsules F8 and F12 was slow and extended over a period of 12 h, and these microcapsules were found suitable for oral controlled-release formulations. The in vitro drug release studies of all formulations are shown in Figures 2–4.

Figure 2

In vitro release of cefdinir from chitosan microcapsules. Each point represents mean ± SD (n = 3)

Figure 4

In vitro release of cefdinir from methyl cellulose microcapsules. Each point represents mean ± SD (n = 3)

Fourier Transform Infrared Spectroscopy

FT-IR spectra of drug, polymers used and the final formulation were taken to study the interaction between them. The results were shown in [Figure 5]. FT-IR spectrum peaks of Cefdinir are observed at 1080, 1180, 1685, 1769, and 3300 cm-1. Alkene groups (=C–H stretching) are seen at 3300 cm-1. The ketone groups (–C=O) are observed at 1685–1769 cm-1. Aromatic (Para) hydrogen bond stretching was observed at above 800 cm-1 that is at 807 cm-1 and 1080 cm-1, and the peaks below 800 cm-1 and 700 cm-1 are aromatic (ortho) hydrogen bond stretching was seen.

Figure 5

FT-IR spectra of (a) cefdinir, (b) sodium alginate, (c) Carbopol 934P, (d) physical mixture of Carbopol 934P microcapsules, and (e) Carbopol 934P microcapsules

DISCUSSION

The prepared microcapsules were found to be discrete, spherical, and free-flowing. The microcapsules were uniform in size, with a size range of 760 ± 8.14 to 986 ± 8.05 μm. The SEM photograph indicates that the microcapsules were smooth and spherical [Figure 1]. From the results of FT-IR, it was clear that there was no interaction between drug and polymers used in formulation [Figure 5].

The microencapsulation efficiency was in the range of 55–92%. Microcapsules with a coat consisting of alginate and a mucoadhesive polymer exhibited good mucoadhesive properties in the ex vivo wash-off test. The wash-off was slow in the case of Carbopol 934P microcapsules with retention of 80% ± 1.35 after 1 h. The wash-off was faster at intestinal pH than at gastric pH in case of Carbopol 934P and methyl cellulose microcapsules. The rapid wash-off of Carbopol 934P microcapsules observed at intestinal pH is due to the ionization of carboxyl and other functional groups in the polymers at this pH,[25] which increases their solubility and reduces adhesive strength; but for chitosan microcapsules the wash-off was faster at gastric pH due to the high solubility of chitosan at acidic pH. From the results of the wash-off test [Table 3], it is clear that the prepared microcapsules had good mucoadhesive properties.

Figure 2 shows the release of cefdinir from the chitosan microcapsules. In all the formulations, the release media were changed after 2 h from simulated gastric fluid at pH 1.2 to a simulated intestinal fluid at pH 6.8 to mimic gastrointestinal transit. The drug release was faster at gastric pH than that of intestinal pH due to high solubility of chitosan at gastric pH. The drug release of all the formulations containing chitosan reached 100% in less than 6 h.

Figure 3 shows release of cefdinir from Carbopol 934P microcapsules. The drug release was less at acidic pH but release was increased at pH 6.8 in case of Carbopol microcapsules and the release was controlled up to 12 h. Figure 4 shows release of cefdinir from methyl cellulose microcapsules and it is clear from the graph that the release was higher at gastric pH and the release of drug was controlled up to 12 h.

Figure 3

In vitro release of cefdinir from Carbopol 934P microcapsules. Each point represents mean ± SD (n = 3)

FT-IR spectrum peaks of cefdinir were observed. By observing the FT-IR spectrum of Carbopol 934P microcapsules, the characteristic peaks with the same wave numbers as that of drug and polymers were observed, that is, there was no merging of peaks with the excipients was seen. As there was no merging of peaks, there was no interaction between the excipients and the drug.

CONCLUSION

The spherical microcapsules with a coat containing alginate and mucoadhesive polymer (chitosan, methylcellulose, or Carbopol 934P) could be prepared by an orifice–ionic gelation process. Among three polymers used in different formulations, Carbopol 934P microcapsules showed good mucoadhesive properties in the ex vivo wash-off test. Cefdinir release from the Carbopol 934P mucoadhesive microcapsules was slow and extended over longer periods of time and depended on the composition of the coat. Drug release was diffusion controlled and followed zero-order kinetics. From the results of FT-IR, it can be concluded that there is no interaction between the drug and the polymers used. The results of SEM show that the formulated microcapsules were smooth and spherical in shape. The results of our study suggest that Carbopol 934P mucoadhesive microcapsules are suitable for oral controlled release of cefdinir.