int j pharm sci nanotech vol 10; issue 1 international journal of...

3582 Int J Pharm Sci Nanotech Vol 10; Issue 1 January February 2017

Research Paper

Development, in vitro and in vivo Evaluations of Solid-Lipid Microparticles based on Solidified Micellar Carrier System for Oral Delivery of Cefepime Chukwuebuka Umeyor1*, Uchechukwu Nnadozie1 and Anthony Attama2 1Nanomedicine and Drug Delivery Research Unit, Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka 422001, Anambra State, Nigeria; and 2Drug Delivery and Nanomedicine Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka 410001, Enugu State, Nigeria.

Received September 20, 2016; accepted December 29, 2016

ABSTRACT

This study seeks to formulate and evaluate a solid lipid nanoparticle-based, solidified micellar carrier system for oral delivery of cefepime. Cefepime has enjoyed a lot of therapeutic usage in the treatment of susceptible bacterial infections; however, its use is limited due to its administration as an injection only with poor patient compliance. Since oral drug administration encourage high patient compliance with resultant effect in improved therapy, cefepime was formulated as solid lipid microparticles for oral delivery using the concept of solidified micellar carrier system. The carrier system was evaluated based on particle yield, particle size and morphology, encapsulation efficiency (EE %), and thermal analysis using differential scanning calorimeter (DSC). Preliminary microbiological studies were done using gram

positive and negative bacteria. In vitro release study was performed using biorelevant media, while in vivo release study was performed in white albino rats. The yield of solid lipid microparticles (SLM) ranged from 84.2 – 98.0 %. The SLM were spherical with size ranges of 3.8 ± 1.2 to 42.0 ± 1.4 μm. The EE % calculated ranged from 83.6 – 94.8 %. Thermal analysis showed that SLM was less crystalline with high potential for drug entrapment. Microbial studies showed that cefepime retained its broad spectrum anti-bacterial activity. In vitro release showed sustained release of cefepime from SLM, and in vivo release study showed high concentration of cefepime released in the plasma of study rats. The study showed that smart engineering of solidified micellar carrier system could be used to improve oral delivery of cefepime.

KEYWORDS: Solid-lipid nanoparticle; Cefepime; Solvent injection; reverse micellar solution.

Introduction

Particulate drug delivery systems (PDDS) especially lipid-based formulations have been shown to enhance the bioavailability of drugs (Umeyor et al., 2013). Lipid-based formulations can be used to influence the absorption of active pharmaceutical ingredients (API) through different mechanisms to modify bioavailability (Fouad et al., 2011). The high interest in the use of lipid-based drug delivery systems in drug formulation and delivery is mainly due to the high versatility and diversity of pharmaceutical grade lipid excipients employed, and their compatibility with solid, semi-solid and liquid dosage forms (Attama and Nkemnele, 2005). Recently, biocompatible and biodegradable lipid microparticles have been reported as potential alternative drug carrier systems to polymers. This is because they provide sustained and controlled delivery of drug for long periods of time. Biodegradable matrix drug delivery system such as solidified reverse micellar delivery systems is a potential drug delivery system for sustained release of hydrophilic and lipophilic drugs.

Solidified reverse micellar solution (SRMS) based on a stabilizer heterolipid, Phospholipon® 90G and solid lipid such as Softisan® 154, a hydrogenated palm oil transform into a lamellar mesophase after melting on contact with water. This mesophasic change imparts controlled release behaviour to entrapped drugs (Friedrich and Muller-Goymann, 2003; Schneeweis and Muller-Goymann, 2000; Chime et al., 2013). Solid lipid microparticulate (SLM) carrier systems are physiologically compatible, physicochemically stable and allow a large scale production at a relatively low production cost. They are also administered through different routes such as oral, topical, ophthalmic, subcutaneous and parenteral (Umeyor et al., 2012; Mishra et al., 2012).

Softisan®154 is hydrogenated palm oil with a melting point of 53 – 58 °C. It is a hard fat based on triglyceride blends of natural, saturated even-numbered vegetable fatty acids with chain length of C10 – C18. Phospholipon®90G contains about 90 % of phosphatidylcholine stabilized with 0.1 % ascorbyl

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 10Issue 1 January – February 2017

MS ID: IJPSN-09-26-16-UMEYOR

3582

Umeyor et al: Development and Evaluations of Solid-Lipid Microparticles System for Oral Cefepime 3583

palmitate and is a safe (GRAS) FDA-approved excipient with applications in drug delivery (Nnamani et al., 2010). Cefepime is a semi-synthetic, fourth-generation cephalosporin developed in 1994. Like other fourth-generation cephalosporins, it has an extended and greater activity against gram-positive and gram-negative bacteria than third generation cephalosporins. It is a reserved antibiotic for severe nosocomial infections caused by multi-resistant microorganisms. It is only available as a dry, reconstitutable powder for injection (Drugspedia, 2016). The therapeutic profile of any drug entity is generally affected by the method of formulation and administration route. Since orally administered drugs boast a high level of patient compliance, it has become very important to seek new and smart ways of improving the delivery of drugs like cefepime which is currently administered parenterally as injections only. Literature lacks any in vitro and in vivo data on the oral delivery of cefepime using any form of lipid based drug delivery system.

Therefore, this study seeks to formulate and carry out in vitro and in vivo evaluations of solid lipid microparticles based on solidified micellar carrier system for oral delivery of cefepime.

Materials and Methods

The materials used were: Phospholipon ®90G (Phos-pholipid GmbH, Koln, Germany), Softisan®154 (Fa. Condea Chemie GmbH, D-58453Witten, Germany), Cefepime (Bharat Parenterals Ltd, India), Ethanol, Acetone (Sigma Aldrich, Germany), Polysorbate (Tween®) 80 (BDH, Poole, England). All other reagents and solvents used were analytical grade. Distilled water was obtained from a glass still.

Preparation of solidified reverse micellar solutions (SRMS)

Mixtures of Phospholipon®90G and Softisan®154 (1:2 % w/w) were prepared by simple fusion. Briefly, each lipid was weighed out using an electronic analytical balance (Adventurer®, Ohaus, China) and melted at 70 °C with stirring in a thermo-regulated water bath until a homogenous, transparent lipid melt was obtained. Stirring continued at 28 ± 1 °C (prevailing room temperature) until the lipid solidified to give the SRMS, which was properly stored.

Preparation of solid lipid microparticulate (SLM) system of cefepime

The cefepime-loaded SLM were prepared using the appropriate amount of ingredients given in Table 1. In each case, a known quantity of SRMS (P90G: S154, 1:2 % w/w) at 70 °C was added to ethanol-acetone (1:2 % v/v) mixture, and a measured amount of cefepime was added into the micellar solution. The aqueous phase was prepared by dispersing 2 mL of polysorbate (Tween® 80) in 30 mL of distilled water and brought to the same temperature as the lipidic phase. With the aid of a 5 mL syringe fitted with a 21G needle, the drug-loaded micellar solution was injected drop-wise into the hot

aqueous phase with stirring, and the mixture was homogenized at 2000 rpm (Stuart®, Bibby Scientific, Staffordshire, UK) for 30 min to produce a hot emulsion. The resulting dispersion obtained was filtered with a filter paper (Whatmann No. 1, Ø = 90 mm, 11 μm) to remove any excess lipid, and the lipid microparticles were allowed to cool at room temperature. The cooled lipid microparticles were dried for 72 h at 28 ± 1 °C using a dessicator with silica gel as a dessicant. This procedure was similar to a reported study (Schubert and Muller-Goymann, 2003). Several batches of the cefepime-loaded SLM were formulated.

TABLE 1

Quantities of starting materials for the formulation of the different batches of unloaded and Cefepime-loaded SLMs.

Batch Cefepime (% w/w)

Lipid base 1:2 (%w/w) (P90G: Softisan 154)

Tween 80 (%w/w)

Distilled water, qs (%w/w)

A1 0.3 15.0 2.0 100.0 A2 0.6 15.0 2.0 100.0 B1 0.5 15.0 2.0 100.0 B2 1.0 15.0 2.0 100.0 C1 1.5 15.0 2.0 100.0 C2 3.0 15.0 2.0 100.0 D1 - 15.0 2.0 100.0 D2 - 15.0 2.0 100.0

a Batches A1 and A2, B1 and B2, C1 and C2 contain Cefepime while D1 and D2 contain no drug. P90®G = Phospholipon 90®G

Determination of percentage yield

The dried SLM were weighed to get the yield of SLM formulated per batch. The percentage (%) yield was calculated using the formula:

Percentage (%) recovery =1

2 3

WW + W

× 100 …..(1)

Where, W1 is the weight of the SLM formulated (g), W2 the weight of the drug added (g) and W3 the weight of the lipids and Tween® 80 (g) used as the starting material.

Morphology and time-resolved particle size analysis

Microparticles weighing 10 mg from each batch were dispersed in distilled water and analyzed using a computerized image analyzer with in-built software for measurement of projected diameters of microparticles corresponding to their particle sizes. The slide was covered with a cover slip and viewed with photo microscope (Hund®, Weltzlar, Germany) attached with an electronic image analyzer (Moticam, China) at a magnification of ×600. Several microparticles were counted (n = 100) and their sizes recorded. A mean of particles sizes was calculated as the representative size for each batch. Photomicrographs showing particle morphologies were also taken. All these were done in time-resolved manner (24 h, 2 weeks and 2 months)

Time-resolved pH analysis

Microparticles weighing 10 mg from each batch were dispersed in 50 mL of simulated gastric fluid (SGF, pH 1.2) without enzyme in a 100-mL beaker. The pH of the dispersion was determined in triplicates using a pH


meter (Jenway 3505, USA). This was done at different time intervals of 24 h, 30 days and 90 days post-formulation. The procedure was repeated for all the batches.

Determination of encapsulation efficiency

The encapsulation efficiency (EE, %) of each formulation was determined 48 h after formulation to ascertain the exact amount of drug entrapped in the batches of microparticles at formulation. 50 mg of each batch of the SLM were dispersed in100 mL of distilled water in a 100-mL volumetric flask. The dispersion was allowed to equilibrate for 72 h at room temperature, shaken and filtered. The filtrate was adequately analyzed for cefepime content spectrophotometrically (Jenway UV 6505, USA) at a pre-determined wavelength of 225 nm, which was repeated in triplicates for each batch. The amount of drug encapsulated in the microparticles was calculated with reference to a standard Beer’s plot for cefepime. EE (%) was calculated using the formula below:

EE (%) =Actual Drug Content

Theoretical Drug Content× 100 …..(2)

Determination of drug loading capacity

The drug loading capacity (DLC) of the SLM was also determined. DLC estimates the ratio between the entrapped API and total weight of the lipids. It was evaluated using the relationship below:

Drug Loading Capacity, DLC (%) = a

l

WW

× 100 …..(3)

Where, Wa is the amount of API entrapped by the lipid and W1 is the weight of lipid added in the formulation.

Thermal analysis

Melting transitions and changes in heat capacity of the SRMS and Cefepime-loaded SLM were determined using a differential scanning calorimeter (Netzsch DSC 204 F1, Germany). 10 mg of each batch was weighed into aluminium pan, hermetically sealed and the thermal behaviour determined in the range 50 – 250 °C, under a 20 mL/min nitrogen flux at a heating rate of 10 °C/min. The baselines were determined using an empty pan, and all the thermograms were baseline corrected.

Anti-bacterial activity

The agar diffusion method was used in this study. Molten nutrient agar (20 mL) was inoculated with 0.1 mL of Staphylococcus aureus broth culture. It was mixed thoroughly, poured into sterile petri-dishes and rotated for even distribution of the organism. The agar plates were allowed to set and a sterile cork borer (8 mm diameter) was used to bore three cups in the seeded agar medium. A 0.01-mL volume of each of the samples was added, respectively, into the different cups in each of the plates using Pasteur pipettes. The plates were allowed to stand at room temperature for 15 min to enable the

samples to diffuse into the medium before incubating at 37 °C for 24 h. The diameter of each inhibition zone was measured in triplicates and the average determined. The procedure above was repeated using Escherichia coli, Salmonella typhi, Bacillus subtilis.

In vitro release study of cefepime-loaded SLM

Beer’s plot for cefepime at different concentrations was made at a wavelength of 300 nm for SGF (pH 1.2) and 255 nm for SIF (pH 7.2), and a modified USP XXII rotating paddle system was employed for the release study. 900 mL of freshly prepared dissolution medium was maintained at 37 ± 1 °C and a dialysis membrane (MWCO = 8,000, Spectra/Por®, Spectrum laboratories, Rancho, USA) used was pre-treated by soaking it in the dissolution medium for 24 h prior to the commencement of each release experiment. In each case, 0.5 g of the formulated SLM was placed in the dialysis membrane containing 2 mL of the dissolution medium, securely tied with a thermo-resistant thread and then immersed in the dissolution medium under agitation provided by the paddle at 100 rpm. At pre-determined time intervals, 5 mL of the dissolution medium was withdrawn and analyzed spectrophotometrically (Jenway UV 6505, USA). For each sample withdrawn, an equivalent volume (5 mL) of the dissolution medium was added back to maintain sink condition throughout the study period. The amount of drug released at each time interval was determined with reference to the standard Beer’s plot for cefepime in each of the dissolution medium used.

Kinetic analysis of in vitro release

The release profiles of the SLM in both SGF and SIF were analyzed to determine the kinetic mechanism(s) of the in vitro release study using three models including first-order equation, Higuchi square root model and Hixson-Crowell cube root model. The model with equation of best fit indicates the release kinetic as follows:

Q = 100 (1 – e-k1t) …..(4) Q = K2 (t)1/2 …..(5)

Q = 1001/3 – K3t …..(6) Where, Q is the release percentage at time, t and K1,

K2 and K3 are the rate constants of first-order, Higuchi and Hixson-Crowell models respectively

In vivo release study

All animal experimental protocols were carried out in accordance with guidelines of the Animal Ethics Committee of the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria and EU Directive 2010/63/EU for animal experiments. White albino rats weighing 200 – 250 g were randomly selected for the experiment and kept in separate cages for 2 weeks to acclimatize. They were allowed free access to food and water throughout the study period. The rats were divided into four groups of five rats each. The cefepime-loaded SLM was reconstituted with distilled water and administered orally to the rats at 2, 3, 4, and 5 mg/kg dose levels, respectively. Prior to SLM


administration, blood was drawn from the retro-orbital venous plexus of the rats. Then, with reference to their body weights, the calculated dose volumes were administered to the rats as follows: A1 – A2 (2 mg/kg), B1 – B2 (3 mg/kg), C1 – C2 (4 mg/kg), while the control group received the unloaded SLM (i.e. D1 – D2) at 5 mg/kg. No positive control was administered to the animals since there is currently no oral formulation of cefepime in the market. After drug administration, blood was drawn from the retro-orbital venous plexus of the rats at 1, 3, 5, 8, 12 and 24 h and introduced into eppendorf tubes. The blood samples were centrifuged at 4000 rpm for 15 min, and their sera carefully collected, diluted immediately with distilled water and analyzed spectrophotometrically (Jenway UV 6505, USA) at 280 nm.

Data and statistical analysis

Statistical analysis was carried out using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA). All experiments were performed in replicates (n = 3) for validity of statistical analysis. Results were expressed as mean ± SD. ANOVA and student t-tests were performed on the data sets and differences were considered significant for p values < 0.05.

Results

Percentage recovery of SLM

The values of the percentage recoveries of SLM post-formulation are shown in Table 2. From the table, batches A1 and A2 gave yields of 86.0 ± 0.02 and 90.18 ± 0.05 % of the SLM. Batches B1 and B2 yielded 87.0 ±

0.08 and 94.0 ± 0.01 % of the SLM while batches C1 and C2 gave the highest yields of 95.0 ± 0.01 and 98.02 ± 0.10 % of the SLM. The lowest yields of the SLM were obtained from batches D1 and D2 (unloaded batches) as 84.3 ± 0.00 and 84.2 ± 0.08 %.

TABLE 2

Percentage recoveries of unloaded and Cefepime-loaded SLMs.

Batch Cefepime hydrochloride (% w/w) Recovery (%)b

A1 0.3 86.00 ± 0.02 A2 0.6 90.18 ± 0.05 B1 0.5 87.00 ± 0.08 B2 1.0 94.00 ± 0.01 C1 1.5 95.00 ± 0.01 C2 3.0 98.02 ± 0.10 D1 - 84.30 ± 0.00 D2 - 84.20 ± 0.08

b Batches A1 and A2, B1 and B2, C1 and C2 contain Ccefepime while D1 and D2 contain no drug. n = 3.

Morphology and time-resolved particle size analysis

The SLM formed were smooth, spherical and relatively stable throughout the study period. Representative photomicrographs from each sub-batch are shown in Fig. 1 and the corresponding particle sizes are shown in Fig. 2. For the drug-loaded formulations, batches A1 and A2 had the lowest particle sizes ranging from 3.8 ± 1.2 to 11.0 ± 0.8 µm respectively. This was followed by batches B1 and B2 which gave particle sizes range of 12.4 ± 0.2 to 22.1 ± 0.5 µm while batches C1 and C2 yielded particle sizes ranging from 30.8 ± 2.1 to 34.8 ± 1.3 µm respectively. The unloaded batches D1 and D2 had the highest particle sizes of 40.0 ± 1.5 and 42.0 ± 1.4 µm respectively.

A1

B1


A2

B2

Fig.1. Photomicrographs of representative batches of solid lipid microparticles (SLM). A1,B1 and C1 contain 0.3, 0.5 and 1.5% w/w of cefepime and D1 is drug-free. Bar represents 20μm.

Fig. 2. Time-resolved particle size analysis of SLM. Analysis was done after 24 h, 2 weeks, and 2 months of storage (n = 3).

Time-resolved pH stability analysis

The pH of the different batches of SLM was evaluated 24 h, 30 days and 90 days post-formulation at 28 ± 1 °C to determine the possibility of variation of pH with time which could be caused by formulation excipients or drug degradation. The result of the pH study is shown in Table 3. From the result, the pH of batch A formulations ranged from 6.30 ± 0.4 to 6.35 ± 0.5 over a period of 90 days while batch B formulations had pH range of 6.43 ± 0.3 to 6.90 ± 0.2 over the same period. Batch C

formulations maintained a pH range of 6.42 ± 0.3 to 6.46 ± 2.2 while batch D formulations gave pH range of 6.60 ± 0.7 to 6.84 ± 1.6 respectively over 90 days.

Encapsulation efficiency (EE%) and drug loading capacity (DLC)

From the results of EE% shown in Table 4, it could be seen that maximum EE% of 94.80 ± 1.0 and 92.60 ± 1.5 were obtained from batches C1 and C2 while minimum EE% of 83.60 ± 0.1 and 76.00 ± 0.4 were obtained from batches A1 and A2 respectively. Similarly, a maximum

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TABLE 3

Average pH

Batch

A1 A2 B1 B2 C1 C2 D1 D2

c Batches A1D2 contain n

TABLE 4

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Batch

A1 A2 B1 B2 C1 C2

d Batches A1 Pharmaceuti

Therm

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16.72 ± 0.0 anained from ba

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6.33 ± 1.3 6.35 ± 0.5 6.43 ± 0.3 6.87 ± 0.8 6.44 ± 0.2 6.46 ± 2.2 6.84 ± 1.6 6.61 ± 0.6

1 and A2, B1 and Bno drug. n = 3

tion Efficiency oaded SLMs.

Encapsulation Efficiency (%)d

83.60 ± 0.1 76.00 ± 0.4 89.00 ± 0.7 86.00 ± 0.2 94.80 ± 1.0 92.60 ± 1.5

and A2, B1 and B2ical Ingredient. n =

mal analysis

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6.31 ± 0.2 6.34 ± 1.0 6.51 ± 0.7 6.90 ± 0.2 6.44 ± 1.5 6.42 ± 0.3 6.84 ± 0.8 6.61 ± 1.1

B2, C1 and C2 con

(%) and Dru

Drug Loadin(mg API/100

16.72 ±15.20 ±29.67 ±28.67 ±63.20 ±61.73 ±

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Fig. 3e. DSC thermogram of C2 SLM containing 3.0 %w/w of cefepime.

Anti-bacterial activity of SLM

The result of the susceptibility of the model microorganisms to the cefepime-loaded SLM is shown in Fig. 4. The result indicates that cefepime entrapped in the S154-P90G lipid matrix produced very significant (p < 0.05) inhibition on the viability of all the test organisms, but S. aureus which was resistant to the inhibitory effect of the API. The inhibitory effect is seen from the high inhibition zone diameter (IZD) produced from the study.

(a)

(b)

(c)

Fig. 4. Anti-bacterial activity of cefepime-entrapped SLM against E. coli (Fig. 4a), S. typhi (Fig. 4b) and B. subtilis (Fig. 4c) after two months of storage (n = 3).

In vitro release study

The result of the in vitro drug release is shown in Fig. 5 (a – f ). The result showed that the use of S154-P90G lipid matrices in the formulations modified the release of cefepime from the SLM producing a sustained release effect. There was no initial burst effect in both SGF (pH 1.2) and SIF (pH 7.2) used in the study and there was very significant (p < 0.05) release of the entrapped drug from the formulations in both media. However, it could be seen that there was a higher drug release in SIF than SGF for all the formulations.

Kinetic analysis of in vitro release

Different kinetic models which describe the mechanism of release of API from a matrix system were used to explain the release kinetics of cefepime from solid-lipid based microparticulate carrier system. The most appropriate model was selected on the basis of goodness-of-fit test. The analysis of the result (Table 5) showed that all the batches predominantly obeyed Higuchi square root model in both media except batches C1 and C2 which obeyed Hixson-Crowell model of release.

Fig. 5a. In vitro release profile of Cefepime from SLM (A1 and A2) loaded with 0.3 and 0.6 % w/w of drug in SGF (n = 3).


Fig. 5b. In vitro release profile of Cefepime from SLM (B1 and B2) loaded with 0.5 and 1.0 % w/w of drug in SGF (n = 3).

Fig. 5c. In vitro release profile of Cefepime from SLM (C1 and C2) loaded with 1.5 and 3.0 % w/w of drug in SGF (n = 3).

Fig. 5d. In vitro release profile of Cefepime from SLM (A1 and A2) loaded with 0.3 and 0.6 % w/w of drug in SIF (n = 3).

Fig. 5e. In vitro release profile of Cefepime from SLM (B1 and B2) loaded with 0.5 and 1.0 % w/w of drug in SIF (n = 3).

Fig. 5f. In vitro release profile of Cefepime from SLM (C1 and C2) loaded with 1.5 and 3.0 % w/w of drug in SIF (n = 3).

TABLE 5

Release kinetics of Cefepime from SLM in SGF and SIF.

Batch First order model (r2)

Higuchi Square root model (r2)

Hixson-Crowell cube root model (r2)

eSGF eSIF SGF SIF SGF SIF A1 0.789 0.685 0.998 0.995 0.992 0.991 A2 0.595 0.780 0.999 0.997 0.995 0.994 B1 0.880 0.880 0.999 0.998 0.998 0.995 B2 0.885 0.685 0.997 0.999 0.981 0.999 C1 0.485 0.590 0.997 0.998 0.999 0.999 C2 0.580 0.750 0.989 0.998 0.999 0.999

eSGF = Simulated Gastric Fluid, SIF = Simulated Intestinal Fluid.

In vivo release of cefepime

The result of the in vivo drug release is shown in Fig. 6 (a – c). The result showed that there was significant (p < 0.05) release of cefepime in the systemic circulation of the animals used in the study following oral administration. Furthermore, there was sustained release of cefepime in all the batches. However, after 24 h of study, a maximum concentration of 1.6 µg/mL of cefepime was released from batches A and C SLM, while batch B SLM released the least concentration


(1.4 µg/mL) of cefepime after 24 h of study. There was no release of cefepime from batch D SLM, since they were not loaded with the drug.

Fig. 6a. In vivo release profile of Cefepime from SLM (A1 and A2) (n = 3).

Fig. 6b. In vivo release profile of Cefepime from SLM (B1 and B2) (n = 3).

Fig. 6c. In vivo release profile of Cefepime from SLM (C1 and C2) (n = 3).

Discussion From Table 2, it could be seen that the recovery of

cefepime-loaded SLM increased with increase in drug loading. The high percentage recoveries could be attributed to the high encapsulation of the drug in the SRMS which was highly solubilized in the lipid matrix. This delayed the precipitation of the lipidic matrix from the organic phase allowing a longer diffusion between the solvent and aqueous phases and resulting in a more effective and complete precipitation of the lipidic matrix in the form of SLM (Doktorovova and Souto, 2009). The variations in particle sizes obtained could be due to the process parameters such as lipid matrix concentration, solvent mixture concentration, and injected amount, the rate of injection of cefepime-loaded lipid matrix and emulsifier concentration. The small particle sizes might be due to the use of polysorbate (Tween®) 80 and phospholipon®90G as emulsifiers in the aqueous and organic phases respectively. Increase in the amount of drug loaded in the formulations significantly (p < 0.05) increased the particles sizes (3 – 11 µm in batches A1 and A2, and 30 – 34 µm in batches C1 and C2). This means that drug loading has significant (p < 0.05) effect on the obtained particle sizes as a result of the increase in the concentration of entrapped cefepime. This is consistent with earlier reports (Umeyor et al., 2012; Nahla and Alaa-Eldeen, 2006). A comparison of cefepime-loaded SLM batches (A1, A2, B1, B2, C1, and C2) with the unloaded batches (D1 and D2) revealed that the unloaded batches produced particles with high particle sizes than the drug-loaded batches. Therefore, since all the batches of SLM formulated contain equal concentration of the lipid matrix, the high particle sizes (40 – 42 µm) observed in batches D1 and D2 could be attributed to increase in viscosity due to high lipid concentration resulting in high emulsion droplets and finally greater microparticle sizes (Mishra et al., 2012). It could also be due to particle coalescence and growth by Ostwald ripening or sintering because it has been shown microscopically, that many disperse systems flocculate shortly after preparation (Attama et al., 2009), though it was not observed in the loaded formulations. It is necessary to have a sound knowledge of the stability profile or the pH of maximum stability of a drug, and use it in the design and formulation of a stable dosage form for the API (Umeyor et al., 2012). Stability study was done in SGF to ascertain the acid stability profile of the drug when formulated as SLM since the formulations were intended for oral delivery. Also, the quoted temperature was the prevailing room temperature at the time of study; its importance was to determine if it would influence the pH profile of the formulations. From Table 3, batches A1 and A2 showed steady decrease in pH over 90 days while batches B1 and B2 showed initial increase in pH after 30 days followed by a decrease in pH after 90 days. In contrast, batches C1, C2, D1 and D2 maintained a somewhat steady pH over 90 days. This observation shows that batches C1, C2, D1 and D2 were more stable than batches A1, A2, B1 and B2. However, all the batches maintained their pH in the acidic region,


implying that SRMS-based SLM technology could be successfully applied for oral delivery of cefepime.

An important property of SLM is their ability to accommodate active molecules (Mishra et al., 2011). From Table 4, it could be seen that drug encapsulation efficiency increased with increase in the concentration of the drug such that the maximum encapsulation efficiency were 94.80 and 92.60 % which are batches C1 and C2 containing 1 % w/w each of drug, whereas the minimum encapsulation efficiency were 83.60 and 76.00 % which are sub-batches A1 and A2 containing 0.3 % w/w each of drug. This showed that both the mobile stabilizer (polysorbate 80) and the stabilizer heterolipid (Phospholipon®90G) with the lipid matrix promoted concentration-dependent drug solubilization. The SRMS accommodated more drug at higher drug loadings due to the low crystalline nature of the excipients. The variation in the values of EE% obtained could be as a result of drug and SRMS physicochemical and materials characteristics. This suggests that high entrapment of cefepime in the formulations could be affected by a possible crystalline rearrangement in the lipid matrix used, since it has been reported that when lipid matrices are prepared with solid lipids structured with Phospholipon®90G, there is disorganization in the crystalline arrangement of the lipid matrices which favours drug localization (Chinaeke et al., 2014). It also showed that there was no formation of mixed micelles. The results of the DLC of the cefepime-loaded SLM is somewhat low compared to the concentration loaded in the formulations. This could be attributed to the hydrophilicity of the API due to the fact that DLC is affected by the degree of lipophilicity of the API, and because the formulations were not lyophilized to obtain dry SLM.

The melting endotherm of Softisan®154 was 59.0 °C with an enthalpy of -8.873 mW/mg (Fig. 3a) This melting point value is different from what is obtained from the product information sheet (53 – 58 °C) probably due to variations in sensitivity and calibration of the analytical instrument. The P90G thermogram (Fig. 3b) showed a melting endotherm of 232.0 °C with an enthalpy of -0.6065 mW/mg showing that P90G is less crystalline with a high tendency to entrap and retain cefepime over time (Nnamani et al., 2010). The single, sharp endothermic melting temperature of P90G suggests that the heterogenous stabilizer used for the formulation is pure and stable. The S154-P90G lipid matrix gave a DSC melting temperature of 65.0 °C with an enthalpy of -2.575 mW/mg (Fig. 3c). This suggests that the lipid matrix is less crystalline than the bulk lipid, which means that an imperfect matrix was generated due to the distortion of the crystalline arrangement of the bulk lipid which favours drug solubilization and entrapment (Jaspart et al., 2005). The use of the lipid matrix in the formulations gave DSC thermograms according to drug loading. It was observed that batch A1 with the least drug loading of 0.3 % w/w gave a melting peak of 65.9 °C with an enthalpy of -25.27 mW/mg while batch C3 with the highest drug loading of 3.0 % w/w gave a melting

peak of 69.6 °C with an enthalpy of -33.84 mW/mg. The low enthalpies recorded indicated that drug loading further reduced the crystalline orientation of the matrix creating more spaces for drug localization (Nnamani et al., 2010).

The anti-bacterial study was done two months post-formulation, and the essence was to establish that cefepime didnot loose its activity after encapsulation in SLM and after a short period of storage. The agar diffusion method employed in this study is based on the diffusion of the entrapped drug (cefepime) through a solidified nutrient agar seeded with model bacterial organisms. The cefepime-loaded SLMs showed significant (p<0.05) inhibition on all the test organisms except S. aureus, according to the concentration of drug loaded in each batch in the following order: C1 > C2 > B1 > B2 > A1 > A2. This also corresponds to the encapsulation efficiency (EE%) recorded for the sub-batches, which showed that batches C1 > C2 > B1 > B2 > A1 > A2. This implies that batches C1 and C2 gave the highest activities (high IZDs) due to their high drug contents (1.5 and 3.0 %w/w), while batches A1 and A2 gave the least activities (low IZDs) due to their low drug contents (0.3 and 0.6 %w/w). This suggests that the concentration of drug in each batch was high enough to yield concentrations that are equal to or above the minimum inhibitory concentrations (MIC) of cefepime needed to cause inhibition to the proliferation of the test organisms. This shows that the entrapped drug interfered with the penicillin binding protein (PBP) activity involved in the final phase of peptidoglycan synthesis in the organisms thereby compromising the integrity of their cell walls. However, it was observed that the formulations were not active on the test S. aureus which could be a methicillin-resistant strain, since cefepime has being shown to be inactive against methicillin-resistant bacteria. Batches D1 and D2 which served as the negative control, had no inhibition since it contained no drug.

The in vitro release study performed on the formulations gave different release profiles for SGF and SIF. In both media, there was sustained release of cefepime from the formulations without initial burst effect due to proper entrapment of the drug in the lipid matrix. The release of cefepime from the formulations depended on the drug concentration. The effect of drug concentration on release profiles of the formulations tended to be predominantly non-linear as batches C1 and C2 with the highest payload produced the minimum drug release while batches A1 and A2 with the lowest payload produced the maximum drug release. Therefore, drug concentration effect on the release profiles could be represented in increasing order of magnitude as follows: C2 < C1 < B2 < B1 < A2 < A1. The reason for this is unclear but could be attributed to the formulations attaining saturation solubility below 1.0 % w/w drug loading. It is important to note that there was higher drug release from the batches in SIF than SGF. For instance, in SIF, batches A1 and A2 produced 64 % and 70 % drug release after 3 h while in SGF, batches A1 and


A2 produced 25 % and 28 % drug release within the same interval. This might be due to the high permeability of the imperfections of the lipid matrix encapsulating the drug by SIF leading to a corresponding high solubilisation and drug release. It is also expected since cefepime is a weak acid, it will partition more in the alkaline phase than the acidic phase leading to high concentration of drug release in the SIF. Therefore, the release of cefepime from the formulations is a good indication that the engineered lipid matrix could be a veritable tool in the formulation of SLM for oral delivery of the API. The result (Table 5) of the analysis of kinetics of in vitro release of the SLM showed that the Higuchi square root model was predominantly obeyed by almost all the batches in both SGF and SIF except batches C1 and C2 which followed the Hixson-Crowell model of release. This is similar to our earlier report (Umeyor et al., 2012). This indicates that the solubilisation and diffusion of cefepime from the microparticulate carrier system leads to possible surface area variations. There is possibility of controlled release of the drug from the carrier system, which could be attributed to the presence of few diffusion pores and channels on the smooth surface of the SLM.

The result of the in vivo release of cefepime as shown in Fig. 6 depicts sustained release of the drug after oral administration. It showed that very significant (p < 0.05) concentration of cefepime was released from all batches of the SLM. Batches A and C SLM produced the maximum (1.6 µg/ml) concentration of drug release while batch B SLM gave the least (1.4 µg/ml) concentration of cefepime released after 24 h. The result showed that the formulation of cefepime as SLM based on SRMS made it possible for cefepime to be released into the systemic circulation of the rats used in the study. This is interesting because the lipophilic surface of the SRMS matrices might have influenced the dissolution of cefepime in the lipid milieu leading to improved release of the drug through the narrow pores of the SRMS matrix. Throughout the study, there was zero mortality of animals recorded, which is an indication that the formulations were not toxic to the biological system of the rats used in the study.

Conclusions

The formulation of solidified micellar carrier system for oral delivery of cefepime has shown many interesting prospects of utilizing the properties of lipid vehicles to modify and/or control the release of drugs. SRMS has shown that it is possible to formulate lipid particulate systems for administration through various routes. SRMS-based SLM loaded with cefepime were successfully formulated by solvent injection method. The various physicochemical characterization methods the formulations were exposed to showed very promising results, suggesting that further structuring and optimization of the process would be very viable. The study has shown that a smart engineering of lipid drug delivery system such as solidified micellar carrier system

could be properly utilized as a viable carrier system for the oral delivery of cefepime. Looking ahead, there is need to determine the cytotoxic profile of the SLM in respect to its interaction with the hepatic enzymes of the rats since micro/nano-particles concentrate more in the liver. Studies on the effects of SLM on cardiac and renal functions of the animals would be encouraged to ensure that these vital organs are not unnecessarily stressed by the lipids used in the formulation. The possibility of surface modification of the microparticles for targeted delivery of cefepime would be explored. For pharmaceutical and formulation scientists in developing countries actively involved in natural raw materials development for drug delivery, there is need to use some of the natural excipients in abundance in their environment to formulate cefepime and determine how and to what extent the natural excipients modulate drug release. They could also carry out a comparative characterization of the formulation with those formulated using synthetic excipients. This is important considering the reality that micro-and nano-based drug delivery is the future of medicine.

Declaration of interest

The authors state no conflict of interests and have not received any funding for the research or in the preparation of this manuscript.

Acknowledgements

We acknowledge the assistance of Phospholipid GmbH, Koln, Germany and Fa. Condea Chemie GmbH, D-58453 Witten, Germany in providing samples of Phospholipon 90®G and Softisan®154 respectively, used in the study.

References Attama AA, Nkemnele MO (2005). In vitro evaluation of drug

release from self micro-emulsifying drug delivery systems using a biodegradable homolipid from Capra hircus. Int J Pharm. 304: 4-10.

Attama AA, Okafor CE, Builders PF, and Okorie O (2009). Formulation and in vitro evaluation of a PEGylated microscopic lipospheres delivery system for ceftriaxone sodium. Drug Deliv. 16(8): 448-457.

Chime SA, Umeyor CE, Onyishi VI, Onunkwo GC, and Attama AA (2013). Analgesic and Micromeritic evaluations of SRMS-based oral lipospheres of Diclofenac Potassium. Indian J Pharm Sci. 75(3): 302-309.

Chinaeke EE, Chime SA, Kenechukwu FC, Muller-Goymann CC, Attama AA, and Okore VC (2014). Formulation of novel artesunate-loaded solid lipid microparticles (SLMs) based on dika wax matrices: in vitro and in vivo evaluations. J. Drug Del. Sci. Tech. 24(1): 69-77.

Doktorovova S, and Souto EB (2009). Nanostructured lipid carrier-based hydrogel formulations for drug delivery: A comprehensive review. Expert Opin Drug Deliv. 6: 165-76.

Fouad EA, El-badry M, Mahrous GM, Alsarra IA, Alashbban Z, and Alanazi FK (2011). In vitro investigation for embedding dextromethorphan in lipids using spray drying. Digest J Nanomat Bio. 6(3): 1129-1139.

Friedrich I, and Muller-Goymann CC (2003). Characterization of solidified reverse micellar solutions (SRMS) and production


development of SRMS-based nanosuspension. Eur J Pharm Biopharm. 56: 111-119.

Jaspart S, Piel G, Delatte L, and Evrard B (2005). Solid lipid microparticles: formulation, preparation, characterization, drug release and applications. Expert Opin. Drug Deliv. 2: 75-87.

Mishra P, Agrawal S, and Gupta D (2012). Solid lipid microparticles gel loaded with herbal extracts for acne treatment. J Pharm Res. 5(1): 104-107.

Mishra S, Suryawanshi R, Chawla V, and Saraf S (2011). Fabrication and characterization of solid lipid microparticles of ketoprofen. Ars Pharm. 52 (1), 12 – 15.

Nahla SB, and Alaa-Eldeen BY (2006). In vitro characterization of carbamazepine-loaded precifac lipospheres. Drug Deliv. 13: 95-104.

Nnamani PO, Attama AA, Ibezim EC, and Adikwu MU (2010). SRMS142-based solid lipid microparticles: Application in oral delivery of glibenclamide to diabetic rats. Eur J Pharm Biopharm. 76: 68-74.

Schneeweis A, and Muller-Goymann CC (2000). Controlled release of solid-reversed micellar solution (SRMS) suppositories containing metoclopramide HCL. Int J Pharm. 196: 193-6.

Schubert MA, and Muller-Goymann CC (2003). Solvent injection as a new approach for manufacturing lipid nanoparticles – evaluation of the method and process parameters. Eur J Pharm Biopharm. 55: 125-131.

Umeyor C, Kenechukwu F, Uronnachi E, Chime S, Reginald-Opara J, and Attama A (2013). Recent Advances in particulate anti-malarial drug delivery systems: A review. Int J Drug Deliv. 5(1): 1-14.

Umeyor CE, Kenechukwu FC, Ogbonna JD, Chime SA, and Attama AA (2012a). Preparation of novel solid lipid microparticles loaded with gentamicin and its evaluation in vitro and in vivo. J Microencapsul, 29(3): 297-307.

Umeyor CE, Kenechukwu FC, Uronnachi EM, Osonwa UE, and Nwakile CD (2012b). Solid Lipid Microparticles (SLMs): An effective lipid based technology for controlled drug delivery. Am J PharmTech Res. 2(6): 1-18.

Address correspondence to: Chukwuebuka Umeyor, Nanomedicine and Drug Delivery Research Unit, Department of Pharmaceutics and Pharmaceutical Technology, Nnamdi Azikiwe University, Awka 422001, Anambra State, Nigeria. Phone: +234-8063299850; E-mail: [email protected]

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