Design and development of cefdinir niosomes for oral delivery

Abstract

Objective:

The aim of the present study was to develop nonionic surfactant based vesicles (niosomes) to improve poor and variable oral bioavailability of cefdinir.

Materials and Methods:

Cefdinir niosomes were formulated by sonication method using varying concentration of surfactant (span 60), with and without soya lecithin, but the cholesterol ratio was kept constant in all the formulations. The influence of formulation variables such as surfactant concentration, soya lecithin presence or absence were optimized for size and entrapment efficiency. Drug excipient interaction studies were performed using FTIR, indicating compatibility of excipients with drug.

Results:

The highest entrapment efficiency (74.56%) was observed when span 60, cefdinir, cholesterol and soya lecithin were used in the ratio of 5:1:1:1. The zeta sizer of the niosomal formulations showed the size range between 190 nm-1140 nm. The photomicrography showed round shape of vesicles and further nano size of niosomes was confirmed by scanning and transmission electron microscopy. The optimized niosomal formulations (F11 and F6) exhibited sustained in-vitro release of 94.91% and 94.07% respectively upto 12 h. The ex-vivo permeation studies of optimized formulation revealed that the niosomal dispersion improved cefdinir permeability across goat intestinal membrane as compared to plain drug solution and marketed suspension (Adcef®). Antimicrobial activity studies revealed that the niosomes potentiated bacteriostatic activity of cefdinir as compared to Adcef®.

Conclusion:

The niosomal formulation could be one of the promising delivery system for cefdinir with improved oral bioavailability and controlled drug release profile.

Cefdinir is an extended-spectrum, oral, third-generation cephalosporin antimicrobial agent.[1] Cefdinir is classified in the Biopharmaceutics Classification Scheme (BCS) as a class IV drug. It is used to treat certain infections caused by bacteria, such as pneumonia, bronchitis, ear infections, sinusitis, pharyngitis, tonsillitis, and skin infections.[2,3] Cefdinir has tablet and suspension formulations available in market but they need to be administered frequently. Cefdinir is an orally active drug with incomplete absorption and low bioavailability (16% to 21%). It has shorter half life of 1.7 ± 0.6 h,[4] so there is always a requirement for development of better oral controlled release formulation of cefdinir.

As the resistance has been developed to many of the antibiotics due to their improper use, thus the remaining ones should be utilized in the best possible manner. This drug is of great utility for highly resistant beta-lactamases and also the class cephalosporins are generally well tolerated. Thus this extensively used antibiotic requires judicious and the best method of its proper utilization so as to avoid resistance against the drug. The conventional micellar systems are known to enhance the solubility of poorly absorbed drugs resulting into improved bioavailability.[5,6] Drug delivery system using colloidal particulate carrier, such as liposomes or niosomes, has distinct advantages over conventional dosage form and micelles because the particles can act as drug containing reservoirs.[7,8] Niosomes are a novel drug delivery system, in which the medication is encapsulated in a vesicle. The vesicle is composed of a bilayer of non-ionic surface active agents and these non-ionic surfactant based vesicles (niosomes) are formed from the self-assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer structures.[9] They have been used to solve the problem of insolubility, instability and rapid degradation of various drugs.

Controlled drug delivery along with better oral bioavailability of the drugs using vesicular systems remains the area of interest in the field of pharmaceuticals. Conventional drug delivery system does not release the drug in a controlled manner due to which fluctuations in drug plasma concentration is observed. The maintenance of therapeutic window for the longer duration of time by administering a single dose can be achieved by niosomal formulation.

Based on this hypothesis encapsulation of cefdinir in vesicular structures like niosomes can be predicted to prolong the existence of the drug in systemic circulation and can increase the bioavailability. Niosome formation of cefdinir can further reduce the associated side effects by reducing the dose and also the niosomes requires economical facilities for formulation and characterization.

Materials and Methods

Materials

Cefdinir was generous sample from Nector Lifesciences, Derabassi, India. Cholesterol, soya lecithin and nutrient agar was obtained from HiMedia Laboratories, Mumbai. Span 60, span 40, potassium dihydrogen phosphate, disodium hydrogen phosphate was purchased from Central Drug House, New Delhi. All other materials and solvents used in this study were of analytical grade.

Methods

Preparation of niosomes

Cefdinir niosomes were prepared using sonication method.[10] The surfactant concentrations used was in the range of 100-600 mg (span 60) whereas cholesterol and soya lecithin concentrations were kept constant to 100 mg. Some formulations were also prepared without soya lecithin. The active substance, cefdinir (100 mg) and all the components were added in phosphate buffer pH 6.8 (5-8 mL). The samples were sonicated for 5 min using a Probe Sonicator (PCI Analytics Pvt. Ltd, Mumbai). The procedure resulted in unilamellar vesicles which were cooled down to 25°C directly after sonication and allowed to stand for 24 h.

Drug excipient interaction studies

Fourier transform infrared spectroscopy (FTIR) of pure drug cefdinir and mixture of drug with excipients (niosomal formulation) was taken using Perkin Elmer FTIR spectrophotometer (RXIFT-IR system, USA). Sample was prepared with potassium bromide and data were collected using FTIR, indicating compatibility of excipients with drug.

Characterization of Cefdinir Niosomes

Drug content

Cefdinir content in the niosomes was estimated by using phosphate buffer pH 6.8 as solvent, by a UV-Vis spectrophotometer (Shimadzu - 1700, Japan) based on the measurement of absorbance at 287 nm.

Entrapment efficiency

Each formulation was centrifuged using centrifuge apparatus (REMI LJ01, Mumbai) at 5000 rpm for 30 min at 25°C (room temperature) to separate the free drug in the supernatant from the drug incorporated in the niosomes. The concentration of the drug in the supernatant was determined mathematically using UV-Vis spectrophotometer at 287 nm. The amount of drug incorporated in the niosomes was calculated from the difference in drug concentrations between the supernatant and the original given concentrations.[11]

The entrapment efficiency was calculated using the following equation:

Determination of vesicle size

Sizing was carried out on Zetasizer 300HSA (Malvern Instruments, Ltd, UK).

Optical microscopy

Photomicrographs were taken with a Nikon Microphot FXA light microscope.

Scanning electron microscopy

The niosomes were observed under a scanning electron microscopy (SEM) (JSM 6100 JEOL, Tokyo, Japan). They were mounted directly onto the SEM sample stub using double-sided sticking tape and coated with gold film of thickness of 200 nm under reduced pressure of 0.001 mmHg. Photographs were taken at suitable magnification.

Transmission electron microscopy

The prepared niosomal formulation was characterized for their morphology using transmission electron microscopy (TEM). Briefly, to an aliquot of a suspension of prepared niosomal formulation, sufficient quantity of 1% phosphotungstic acid was added and mixed gently. A drop of the mixture was placed on to the carbon-coated grid and drained off the excess. The grid was allowed to dry, and it was observed under TEM (Hitachi H7500, Japan). Photographs were taken at suitable magnification.

In vitro release studies

In vitro release of cefdinir from the niosomes was studied in both 0.1 N HCl and phosphate buffer pH 6.8 for 12 h each using United States Pharmacopeia (USP) type II Paddle type apparatus[12,13] (Electrolabs, India) using volume 900 mL, at 100 rpm and 37°C. Samples (5 mL) were withdrawn through pipette at different time intervals and were assayed at 287 nm for cefdinir content spectrophotometerically.

Kinetic modeling

The in vitro release of drug is mainly assessed by fitting the cumulative drug release into the kinetic models. This was calculated from the slope of the linear steady portion of % cumulative amount released vs. time (h) plot. The release data obtained were fitted to zero order, first order, Higuchi, Korsmeyer-Peppas kinetic models to determine the mechanism of drug release from the various batches. The correlation coefficients (r²) for the different drug release kinetic models were determined.

Ex vivo permeation studies

Ex vivo permeation of cefdinir from the niosomes was studied in 900 mL of 0.1 N HCl for the first 2 h and in 900 mL of phosphate buffer pH 6.8 for the next 10 h by everted sac method (using goat intestinal membrane) on a magnetic hot plate, with continuous stirring using magnetic bead and 37°C was maintained throughout the experiment.[14] The goat intestinal membrane was everted inside out, washed thrice with buffer pH 6.8 and kept for 12 h in the fresh buffer solution (pH 6.8) before use. One end was sealed using thread and a tube was inserted in sac. Samples (5 mL) were withdrawn at different time intervals and were assayed at 287 nm for cefdinir content using spectrophotometer.

Antimicrobial Activity

The optimized formulation (F11) was assessed for antimicrobial/bacteriostatic activity using ditch plate technique. Staphylococcus aureus strain was used as an indicator. The culture medium selected for this purpose was Nutrient Agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India) and the pH of 7-7.5 was maintained to retard the growth of unlike organisms. The medium was sterilized using an autoclave at 121°C for 20 min. Staphylococcus aureus culture was prepared for inoculum preparation. Petri dishes (9 cm-diameter) containing medium to a depth of 5 mm were used. The inoculum (0.5 mL) was spread over the surface of media and after appropriate solidification (37 ± 2°C for 10 min), niosomal formulation, drug solution and marketed formulation (each equivalent to 1 mg of cefdinir) were applied. Laminar air flow was used throughout the experiment to maintain sterility. Finally, the petri dishes were incubated at 37 ± 2°C for 24 h in reverse position. The zone of inhibition (mm of diameter) was calculated.[15]

Stability of cefdinir niosomes

The prepared formulations were tested for stability by storing them at 4 ± 1°C and at 25 ± 2°C. Residual drug content was assessed on 45^th and 90^th day.[16] The shape of the vesicle was observed under light microscope.

Statistical approach

Graph Pad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. The experiments were performed in triplicate. Results are expressed as the arithmetic mean ± standard deviation (SD).

Results

Preparation and characterization of niosomes

The cefdinir niosomes were prepared by sonication method using span 60, cholesterol and soya lecithin. Niosomes could not be prepared by thin film hydration and ether injection method due to insolubility of cefdinir in most of the organic solvents. The prepared niosomes were evaluated for drug content, entrapment efficiency, vesicle size, optical microscopy, scanning electron microscopy, transmission electron microscopy, in vitro drug release, ex vivo permeation studies and interaction between drug and excipients by FTIR.

Formulations were prepared by varying surfactant (span 60) compositions with and without soya lecithin. Phosphate buffer pH 6.8 was used as the hydration medium for the preparation. The highest entrapment efficiency of 74.56% was observed when span 60, cefdinir, cholesterol and soya lecithin were used in the ratio of 5:1:1:1 (F11) while the lowest was observed to be 42.96% (F7). The size of the vesicles was determined using Zetasizer. The niosomes with size above 1 μm were observed in case of some niosomal formulations (F2, F3, F4, F8) and the least vesicle size (192 nm) was observed in F6 formulation which may be due to change in concentration of span 60 [Figure 1]. Cefdinir content in the niosomes was estimated by UV-Vis spectrophotometer at 287 nm. The highest drug content in the niosomes was 95.98% (F11) and the lowest was 89.43% (F9) [Table 1], thus the formulations had the optimum and uniform drug content. Based upon the entrapment, drug content and size, the niosomal formulations were selected and further evaluated.

Figure 1.

Particle size distribution of niosomal formulation (a) F11 (b) F6

Table 1.

Composition and characterization of niosome formulation

Photomicrographs [Figure 2] taken with a light microscope gives the idea of shape attained by the vesicles. The prepared niosomal formulation was characterized for morphology using SEM and TEM. The SEM provided crystal structure/orientation. SEM of formulation F11 showed the smooth surface of niosomes formed [Figure 3]. Some unevenness of vesicles that observed under the study may be due to drying process under normal environment condition. TEM microphotographs gave sub nanometer resolution of the observed (F11) formulation. Niosomal vesicles appeared as spherical and multilamellar under TEM [Figure 4]. It also revealed that there was no structural deformation thus accessed the stability of niosomal formulation.

Figure 2.

Photomicrograph of niosome formulation (F11)

Figure 3.

SEM microphotograph of (F11) niosome preparation

Figure 4.

TEM microphotograph of (F11) niosome preparation

Drug excipient interaction studies

FTIR of cefdinir and mixture of drug with excipients suggested that there was no interaction between the drug and excipients used. The FTIR spectrum peaks of cefdinir were observed at 1096, 1170, 1660, 1730 and 3390 cm⁻¹. Alkene groups (=C-H stretching) were seen at 3390 cm⁻¹. The ketone groups (-C = O) were observed at 1660-1730 cm⁻¹. Aromatic (Para) hydrogen bond stretching was observed at above 900 cm⁻¹ i.e., at 986 cm⁻¹ and 1096 cm⁻¹, and in the peaks below 800 cm⁻¹ and 700 cm⁻¹ aromatic (ortho) hydrogen bond stretching was seen. Same peaks were obtained from FTIR scan of niosomal formulation (F11), so this confirmed the compatibility of drug with excipients [Figure 5].

Figure 5.

FTIR spectra of (a) cefdinir (b) cefdinir with cholesterol, span 60, soya lecithin (in the ratio 1:1:5:1)

In vitro Release Studies

In vitro release in 0.1 N HCl

The drug release from niosomes was found to be sustained. The niosomal formulation F11 showed the maximum cumulative drug release of 84.16% after 12 h while the lowest was 54.9% (F1). The reason for prolonged and high release by the niosomal formulation F11 may be due to soya lecithin as it has multi-functional surface-active properties. It would have prevented agglomeration and kept the drug molecules dispersed. Also, the cholesterol presence built and maintained membranes as reported in earlier studies.[17] Thus, it confirmed the sustained release profile of the niosomal formulations in the acidic condition by oral route.

In vitro release in buffer pH 6.8

The in vitro release of the prepared niosomal formulations in buffer pH 6.8 showed maximum release of 94.91% (F11) after the time period of 12 h and the lowest release observed was 81% (F3). As the drug is acidic, so it is more soluble in basic buffer, which might have made release high in buffer of pH 6.8 than buffer of pH 1.2. Furthermore, the same formulation had shown the highest release in 0.1 N HCl. It may be concluded that the optimum surfactant concentration, presence of cholesterol and soya lecithin would have contributed to it. The in vitro release profile of formulations in buffer pH 6.8 again confirmed sustained release.

Correlation between in vitro release of 0.1 N HCl and buffer pH 6.8

The in vitro release studies were performed in both the mediums separately so as to assess the effect of acidic, as well as the alkaline environment of gastrointestinal track on the prepared niosomal formulations. The release shown by all the prepared niosomal formulations in both the mediums was between 54.9% to 94.91%. The release shown by F11 in buffer pH 6.8 was the best (94.91%) and the difference of release between two release medium was significant (P < 0.05, t-test), release profile decreased in case of 0.1N HCl. The release profile in buffer pH 6.8 also suggests that cefdinir would be best absorbed from the duodenum and jejunum as the upper part of the intestine has same pH 6.8. On these bases the formulations F6 and F11 were concluded as the optimized formulations and were subjected to further evaluation.

Effect of soya lecithin on release

Soya lecithin has several properties like solubilization, crystallization control and an antioxidant activity, as a result all the formulations prepared using soya lecithin (F6-F12) showed better release profile and also the best release profile was observed with soya lecithin formulation (F11). This may be due to the wetting/instantizing property of lecithin that helped powers to dissolve and allowed the mixing of otherwise immiscible substances.[18] It may be concluded that soya lecithin presence contributed for suitable release profile of the niosomal formulations.

Release kinetics

In vitro release data of niosomal formulations from both dissolution mediums were fitted to different equations and kinetic models to explain the release kinetics of cefdinir from niosomes.

The mechanism of drug release from the developed formulations is that most of the formulations in case of release kinetics of niosomal formulation after in vitro release in 0.1 N HCL followed higuchi model r². Both the formulations F6 and F11 followed first release order kinetics and F7 which followed zero order kinetics. In case of release kinetics of niosomal formulation after in vitro release in buffer of pH 6.8 again most of the formulations followed higuchi model r² and both F6, F7 showed zero release order whereas F11 in this followed first order kinetics. The release exponent (n) value for all the formulations was less than 1, i.e., between 0.5-1 indicating anomalous transport. It is generally assumed that the anomalous component of transport is generated by turbulence driven by micro-instabilities. Anomalous diffusion resulted from processes in which particles moved coherently for long times with infrequent changes of direction.[19,20] Thus the kinetic modelling data [Table 2] revealed that the niosomal formulations provided sustained drug action for an extended period of time.

Table 2.

Release kinetics of niosomal formulations after in-vitro release in 0.1N HCl and buffer of pH 6.8

Comparison of optimized formulations with marketed and drug solution by in vitro release

The optimized formulations (F6, F11) were compared with marketed oral suspension (Adcef^®, Torrent Pharmaceuticals, H.P, India) and drug in solution for the in vitro release using buffer pH 6.8 as the dissolution medium. The extended release of 12 h was observed in case of the niosomal formulations as compared to 10 h in case of Adcef^®. The best release profile was shown by F11. The comparative study data revealed the sustained action by the niosomal formulations and percentage release of both niosomal formulations (F6, F11) remained more than that shown by marketed, as well as drug solution [Figure 6].

Figure 6.

Comparison of release profile of optimized niosomal formulations (F6, F11) with marketed (Adcef®) and drug solution

Ex vivo permeation studies

Ex vivo permeation of cefdinir from the niosomes was studied in buffer pH 6.8 using everted sac method to find out the permeation across the gastrointestinal membrane. The optimized niosomal formulations (F6, F11) were selected for ex vivo studies based upon the results from in vitro release and entrapment efficiency. The maximum cumulative % permeation of 74.06% was observed with F11. The pure drug in solution exhibited the least permeation of 60.17%. Thus, the order of permeation potential of different formulations was F11 > F6 > Adcef^® >Drug solution. Ex vivo studies revealed the better permeation potential of niosomal formulations across the intestinal membrane, which again suggests its better oral absorption [Figure 7].

Figure 7.

Ex vivo permeation studies across the goat intestinal membrane

Antimicrobial activity

The results obtained from antimicrobial activity studies assessed the better potential of niosomal formulation of cefdinir in inhibiting the growth of Staphylococcus aureus as compared to marketed formulation (Adcef^®). The values obtained from zone inhibition of niosomal formulation, marketed formulation and drug solution were 31.2 ± 0.87 mm, 27.0 ± 0.42 mm and 25.5 ± 0.02 mm, respectively. The enhanced antimicrobial activity of cefdinir might be attributed to greater drug permeation through niosomal vesicles. The results indicated that the niosomes potentiated antimicrobial activity of cefdinir, thus were suitable candidate for drug delivery [Figure 8].

Figure 8.

Microbiological activity of different formulations using Staphylococcus aureus as test organism

Stability of cefdinir niosomes

The formulations F6 and F11 were tested by storing them at 4 ± 1°C and at 25 ± 2°C. In comparison of formulation F6, the formulation F11 showed better stability as reduction in number of vesicles was found to be less. Storage stability was also evaluated in terms of percent residual drug remaining in the vesicle, considering initially drug content as 100%. A marked reduction in the residual drug content was found when formulations were stored at 25 ± 2°C. The stored cefdinir niosomal formulation was also looked for the shape of the vesicles using photomicrography [Figure 9]. Most of the vesicles were found to be round though some were oval in shape. The maximum drug content after storing at 4 ± 1°C for 90 days was 94.6% (F11).

Figure 9.

Photomicrograph of niosomes (F11) after storage period of 90 days at 4 ± 1°C

Discussion

Niosomes were prepared by sonication method. FTIR of cefdinir and its mixture with excipients confirmed compatibility between the drug and excipients used. SEM and TEM revealed that niosomes were spherical in shape. Niosomes prepared using soya lecithin exhibited higher entrapment efficiency than those prepared without it. The prepared niosomes were assessed for entrapment efficiency in order to calculate the amount of cefdinir entrapped in the vesicles. Entrapment efficiency was calculated based upon the separation of entrapped drug from unentrapped drug. The entrapment was thus found to be in the range 74.56-42.96%. The niosomal formulations prepared using soya lecithin showed higher entrapment in comparison to those prepared without it. It may be concluded that the presence of soya lecithin provided higher entrapment efficiency. Physicochemical properties of drug might have well correlated with the hydrophilic-lipophilic balance of span 60. As cefdinir associates with lipid bilayers, entrapment efficiency depends upon bilayer formation, which might be high in niosomes prepared using span 60. The vesicle size was determined by dynamic light scattering method using particle size analyzer. It has been observed that with increase in the concentration of surfactant from 100-600 mg the size of the niosome first increased (1140 nm) up to 400 mg and later decreased to 823 nm on increasing the concentration of span 60 to 500 mg. Furthermore, increase in the concentration drastically falls back the particle size to 192 nm. Therefore it is because of increase in concentration of surfactant (span 60), which reduces the particle size. In vitro drug release was shown to be retarded by entrapment of cefdinir in niosomes. Soya lecithin formulation showed highest retarded release possibly because it has higher lipophilicity. The in vitro release of drug was assessed by fitting the cumulative drug release into release kinetic models. The formulations followed first order kinetics. The release shown by the niosomal preparations F11 was extended over the period of 2 h and was optimum in overall performance in comparison to marketed preparation (Adcef^®). Ex vivo studies showed significant permeation across the goat intestinal membrane. The permeation potential of niosomal formulations remained high in comparison to marketed preparation which again suggests its better oral absorption. Antimicrobial studies using Staphylococcus aureus revealed that cefdinir niosomal formulation enhanced microbiological activity and thus had better therapeutic activity. The antimicrobial activity of F11 was significantly higher (P < 0.05, t-test) in comparison to Adcef^®. Higher the antimicrobial activity more is the inhibition of bacterial growth of Staphlococcus aureus and more preventing the infection.

During storage, drug leakage and loss in number of vesicles were observed. Lipid vesicles are self assembles of amphiphiles into closed bilayers structures. Hydrated bilayer vesicles, however, are not considered to be thermodynamically stable and are thought to represent a metastable state in that the vesicles possess excess of energy bilayer phospholipids, which can undergo chemical degradation such as oxidation and hydrolysis. Due to this change, vesicular systems maintained in aqueous dispersion may aggregate/fuse, and encapsulated bioactive material may tend to leak out from the bilayer structure during storage. The highest stability profile of F11 might be attributed to the membrane-stabilizing effect of cholesterol and soya lecithin combination.[21,22] The loss of vesicles could be attributed to the disruption/aggregation of vesicles. At 4 ± 1°C, a minimum loss of drug was observed, which might be attributed to the regidization of the vesicles at low temperature that reduced the permeability of the drug through the membrane. Thus, from the results obtained, it can be concluded that prepared vesicular systems are more stable at 4 ± 1°C, as compared to storage at 25 ± 2°C in terms of residual drug content.

Conclusion

The in vitro release, ex vivo permeation studies indicated potential of developed cefdinir entrapped niosomal formulations for better absorption and sustained drug action. The antimicrobial activity studies revealed that the niosomal formulation is more active against Staphylococcus aureus as compared to marketed formulation (Adcef^®). The developed system had also shown potential of maintaining higher level of cefdinir for a longer period of time as compared to plain drug solution and marketed formulation, which suggested the better release profile of the prepared niosomes. From the present study, it was concluded that niosomes can be used efficiently for enhancing absorption and for sustained delivery of cefdinir via oral route.