2 EFFECT OF PROCEESING VARIABLES OFX LS

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Current Drug Delivery, 2013, 10, 517-526 517

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Effect of Processing Variables on Characterization of Ofloxacin Loaded Lipospheres Prepared by Melt Dispersion Technique

Satheesh Babu Natarajan1* and Prabakaran Lakshmanan2

1Karpagam University, Coimbatore, India;

1Faculty of Pharmacy, Lincoln University College, Kuala Lumpur,

Malaysia; 2Asst. Professor, Department of Pharmaceutics, Asia Metropolitan University, Cheras, Malaysia

Abstract: A melt dispersion technique was employed to prepare ofloxacin lipospheres, by using cetyl alcohol (polar lipid). Effects of various process parameters such as selection of surfactants (gelatin, Tween 40 and poly vinyl alcohol) and selection of stirring speed were studied. Lipospheres were evaluated for morphology, drug entrapment and in vitro drug release profiles. The optimized liposphere batch was selected and formulated as tablets and evaluated the in vitro drug release profile. These studies showed that ofloxacin loaded lipospheres were able to control the drug release for a pe-riod of 16h.

Keywords: Lipospheres, ofloxacin, melt dispersion, cetyl alcohol, gelatin, entrapment efficiency, drug release.

INTRODUCTION

The goal of lipid based formulation is to improve the bioavailability of water insoluble drug to an extent greater than that achievable with conventional dosage form. The primary mechanism by which lipid-based formulations en-hance bioavailability is through solubilization of the drug, although other mechanisms of absorption enhancement have been implicated and include reduction of P-glycoprotein-mediated efflux, mitigation of hepatic first pass metabolism through enhanced lymphatic transport [1-3], prolongation of gastrointestinal (GI) transit time, or protection from degrada-tion in the GI tract. Lipid-based formulations have been util-ized to improve the stability of moisture sensitive drugs [4].

Lipospheres, (LS) have been developed as a new type of lipid-based encapsulation system for drug delivery for lipo-philic drugs. LS consist of solid particles with a mean diame-ter usually between 0.2 and 500 m, composed of a solid lipid matrix in which the bioactives are dissolved or dis-persed [5-7]. LS have more advantages over other drug de-livery systems, which include excellent physical stability, low cost of ingredients, ease of preparation and scale-up, and high entrapment of lipophilic drugs [8-15].

The melt dispersion technique has been used successfully in the preparation of lipospheres made from several lipid components. In the present investigation, the ofloxacin drug was incorporated in the lipid matrix system by melt disper-sion technique on lipospheres characteristics, such as drug encapsulation efficiency, shape, and in vitro drug release rate. Besides developing a liposphere- based controlled drug delivery system of ofloxacin, another important objective was to determine the influence of process variables such as the selection of surfactant and stirring speed etc., on

*Address correspondence to this author at the Research scholar, Karpagam University, Echanari Post, Coimbatore, India; E-mail: [email protected]

lipospheres characteristics. Finally the optimized batch ofloxacin loaded lipospheres were prepared as tablets and evaluated in vitro release profiles.

MATERIAL AND METHODS

Materials

Ofloxacin procured from Micro Labs Ltd, Bangalore, India. Cetyl alcohol, Gelatin, Tween 40, PVA was purchased from Loba Chemie Pvt Ltd, Mumbai. All other chemicals used were of analytical grade.

Preparation of Ofloxacin Loaded Lipospheres

Ofloxacin (OFX) loaded liposphere was prepared by melt dispersion technique. At first, the lipidic mixture was melted at 70°C; in which bioactive compound ofloxacin was incor-porated. The melted mixture was emulsified into an external hot aqueous phase containing a suitable surfactant. The o/w emulsion was heated to the same temperature as the melted lipidic phase with continuous agitation using a mechanical stirrer. The hot emulsion was then rapidly cooled to about 20°C by immersing the formulation flask in a cool ice bath without stopping the agitation to yield a uniform dispersion of lipospheres. The obtained LS were washed with distilled water and isolated by filtration. They were allowed to dry at room temperature. All batches were prepared in triplicate (Table 1).

Effect of Formulation and Processing Variables

The effect of the following processing variables on the characterization of microspheres was investigated.

Selection of Optimum Surfactant

Different types of surfactants Tween 40, gelatin and polyvinyl alcohol were selected to stabilize OFX loaded

518 Current Drug Delivery, 2013, Vol. 10, No. 5 Natarajan and Lakshmanan

lipospheres. The prepared lipospheres were evaluated on recovery, drug entrapment and in vitro drug release.

Effect of Stirring Speed

Manufacturing parameters (stirring speed and surfactant) influence the morphology of the particles [13]. Lipospheres of ofloxacin were prepared at different stirring speeds (250, 500, 750 and 1000 rpm) and the effect on encapsulation effi-ciency, morphology and in vitro release studies was investi-gated.

Characterization of OFX Lipospheres

Particle Size and Morphological Analysis

The average particle size of different batches of lipo-spheres was determined by optical microscopy (MEDIC, L-101). The optical microscope was fitted with an eye piece micrometer which was then calibrated with a stage microme-ter. The size of about 100 numbers of particles was measured from each batch and the average size was calculated. The morphology of LS was evaluated by scanning electron mi-croscopy (SEM) observations (HITACHI Model S- 3000H). Samples were mounted on aluminium stubs and coated with gold using a vacuum evaporator. Samples were then exam-ined with a SEM microscope at an accelerating voltage of 10 kV.

Percentage Yield of Lipospheres

The percentage yield of lipospheres (% w/w) was calcu-lated as lipospheres versus the total amount of the drug (ofloxacin) and the excipients added during the preparation:

Yield (%) =WLS+ drug

W Total qty Drug +W Total qty Excipients

100

Determination of Entrapment Efficiency

We aimed to target the drug release through intestinal lymphatic and also the solubility of ofloxacin lipospheres was higher at phosphate buffer pH 6.4. Therefore, the en-trapment efficiency of the OFX loaded lipospheres was de-termined by dissolving 100 mg of each finely crushed lipo-spheres in 100 ml of pH 6.4 phosphate buffer. The obtained solutions were exposed to ultrasonic treatment for 30 min and centrifuged at 2000 rpm for 15 min; the supernatant were analyzed by UV spectrophotometrically at 294 nm. The drug entrapment was determined for each batch in triplicate. The results were shown in the Table 3.

Compatibility Studies

The spectral analysis was done using FT-IR (SHIMA-DZU 8400 SCCE). The dry sample of ofloxacin, cetyl alco-hol, polyvinyl alcohol and physical mixture was mixed by triturating with dry potassium bromide and placed in sample holder.

X-Ray Diffraction Studies [13,14]

X-ray diffraction measurement was performed by X-ray diffraction (XRD, 2 Theta 10–80 ) with copper anode (Cu K

radiation, 40 kV, 30 mA, = 1.54060 Å), using X’celerator as a detector (PANalytical, X’ per PRO). The sample was mounted into a specific device before the measurement by XRD. The data used were typically collected with a step width of 0.0170 and a count time of 15.5 s. The graph was plotted in 2 theta angle Vs intensity count.

Micromeretic Properties of Liposphere

The optimized lipospheres were (TP4-1, TP4-2, TP4-3 and TP4-4) evaluated for flow properties including angle of

Table 1. Composition of Ofloxacin Loaded Lipospheres.

Surfactant (% w/v) Batch Code

Drug: Lipid (w/w)

(Ofloxacin: cetyl alcohol) Gelatin Tween- 40 PVA

Descriptiona

G 1 1: 5 0.025 - - VE

G 2 1: 5 0.050 - - VE

G 3 1: 5 0.075 - - VE

G 4 1: 5 0.100 - - VE

T 1 1: 5 - 0.025 - FF

T 2 1: 5 - 0.050 - FF

T 3 1: 5 - 0.075 - FF

T 4 1: 5 - 0.100 - FF

P 1 1: 5 - - 0.025 SF

P 2 1: 5 - - 0.050 SF

P 3 1: 5 - - 0.075 SF

P 4 1: 5 - - 0.100 SF

a VE: Viscous emulsion; FF: Foams formed; SF: spheres formed at the stirring rate of 250 rpm.

Effect of Processing Variables on Characterization of Ofloxacin Loaded Lipospheres Current Drug Delivery, 2013, Vol. 10, No. 5 519

repose, tapped density, Carr’s compressibility index and hausner ratio [14].

Preparation of OFX Lipospheres Tablet

The optimized OFX lipospheres (TP4-1, TP4-2, TP4-3 and TP4-4) were compressed as per direct compression tech-nique (1000 kg compression force) using single punch tablet machine (SAKTI- India) into tablets using microcrystalline cellulose, cross linked PVP and Polyvinyl pyrollidone (PVP) was used as diluent, disintegrating agent and binder respec-tively. Magnesium stearate was used as a lubricant and aerosil as flow promoter. Then, 220 mg of each mixture was weighed and fed manually into the die of a single punch ta-bleting machine equipped with flat faced punches (11.0 mm) to produce the desired tablets (Table 2).

RESULTS AND DISCUSSION

LS Prepared by Melt Dispersion Technique

The preliminary studies, different drug to lipid ratios (1:1, 1:2, 1:3 1:4 and 1:5), were utilized to prepare ofloxacin lipospheres. On the basis of higher encapsulation efficiency, the lipospheres were prepared by the melt dispersion tech-nique using ofloxacin and cetyl alcohol at 1:5 ratio and the emulsion was stirred at rate of 250 rpm speed. The selection of preparation and processing variables plays a crucial role on the morphology of the particles and entrapment of drug in to the lipid matrix. The present study was aimed to achieve maximum entrapment of ofloxacin and controlled drug re-lease from the lipospheres using a suitable processing condi-tion (Table 1).

Characterization of OFX Loaded Lipospheres

The results of the influence of preparation parameters on the characteristics of the OFX lipospheres are shown in Ta-ble 3. The present work aimed to investigate the effects of the emulsifiers and stirring speed on the fabrication and property of the OFX loaded lipospheres. The Tween 40 (0.025, 0.05, 0.075 and 0.10% w/v) was employed for the preparation of lipospheres. The results revealed that different concentrations of Tween 40, lipospheres obtained possessed poor sphericity and aggregation. In preparation of lipo-spheres by adding gelatin as surfactant, the achievement of smooth spherical particles was found very difficult due to formation of coalescence. An improvement of LS features was obtained in terms of recovery, particles size and aggre-gate formation by of the PVA used as a stabilizer. The mor-phology of the lipospheres was not significantly affected by adding PVA. The encapsulation efficiency of OFX in the lipospheres was only 55% and 62% when PVA at 0.025% to 0.05% respectively. And it was greatly increased to 68% for 0.075% and 69% for 0.10% of PVA.

The effect of stirring rate on the physical characteristics of the microspheres was examined for batch P2, P3 and P4. Both shape and size distribution of the particles depended heavily on the stirring speed (Table 4). Rapid rotation of the stirrer (1000 rpm; batch P4-4) resulted in smaller particle size (48 m), with narrower particle size distribution ob-tained. However, complete drug release (98% after 16 h) was observed from the lipospheres prepared at higher speed. This was presumably due to the small particle size that leads to a much larger surface area available for release. Encapsulation efficiency at 250 rpm speed was low also found to be lower, i.e., 70% than that obtained by stirring speed of 500-750rpm

Table 2. Composition of Tableted OFX Lipospheres.

Ingredients (%, w/w) TP4-1 TP4-2 TP4- 3 TP4-4

OFX lipospheres (mg equivalent) 100mg 100mg 100mg 100mg

Avicel PH 301(mg) 100mg 100mg 100mg 100mg

Polyvinyl pyrollidone (K30) 12mg 12mg 12mg 12mg

Cross polyvinyl pyrollidone 6mg 6gm 6mg 6mg

Magnesium stearate 1mg 1mg 1mg 1mg

Aerosil 1mg 1mg 1mg 1mg

Tablet weight 220mg 220mg 220mg 220mg

Table 3. Effect of Surfactant at Different Concentrations (0.025, 0.05, 0.075 and 0.1% w/v of PVA) on Lipospheres Characteristics.

Batch Mean Diameter ( m) ± SD Yield (%) ±SD Entrapment Efficiency (%) ±SD

P 1 122±1.22 32±1.02 55±1.09

P 2 107±0.84 44±1.87 62±1.33

P 3 99±0.91 48±1.05 68±1.73

P 4 92±0.28 56±1.74 69±1.95


(batch P4-2 and P4-3) which may be due to the increase in effective surface area that led to drug exposure to the solvent during the washing stage. However, agglomeration was ob-served when stirring speed was kept low (250-500 rpm). Finally, the 1000 rpm stirring speed was selected for prepa-ration of lipospheres, at which encapsulation efficiency was found to be 85% and particle size was 48 m.

FT-IR Analysis

The FT-IR spectra of pure drug (ofloxacin), cetyl alcohol, poly vinyl alcohol and physical mixture were taken by pre-paring KBr pellets and analyzed in the scanning Range of 4000 to 500 cm. Fig. (6) shows IR spectra for ofloxacin, ce-tyl alcohol, poly vinyl alcohol and physical mixture (contain-

ing ofloxacin, cetyl alcohol and PVA). From this data (Fig. 6) we found that there is no interaction between physical mixture.

X-Ray Diffraction

X-Ray diffraction is a means of identifying crystalline compounds. It can be particularly useful when these com-pounds are very fine-grained components or mixtures. The X-Ray spectrum of ofloxacin, cetyl alcohol and Formula-tions were determined using X-Ray Diffractometer. The phase compositions of the samples prepared at different pa-rameters were similar due to both the precursor slurry and the drying temperature and were controlled at identical con-dition. Best formulation batch was selected to do the XRD

Fig. (1a). Photomicrograph images of OFX lipospheres prepared by Tween 40 and gelatin (at 0.1%w/v) respectively.

Fig. (1b). Photomicrograph images of OFX loaded lipospheres using PVA as emulsifier (at 0.1%w/v) during emulsification and solidifica-tion stage respectively.

Fig. (2). Scanning electron micrograph images showing the morphology of OFX loaded LS prepared by 0.1% w/v of PVA used as a surfac-tant. (1000x and 100x magnification respectively).


test, as shown in (Fig. 7). The XRD scan of plain ofloxacin showed intense peaks of crystallinity. Diffractogram of ofloxacin showed high intensity peaks between 2 of 20-30

0

values demonstrating the crystalline nature of drug. No in-tense peaks were observed in diffractogram of cetyl alcohol which indicates amorphous nature. The XRD pattern of for-mulation exhibited halo pattern with less intense and denser peaks compared to plain ofloxacin.

In- Vitro Drug Release of OFX Loaded Lipospheres

The release of OFX from lipospheres prepared by melt dispersion technique is shown in (Fig. 8). The drug release from the lipospheres is drug entrapment dependent, the

higher lipid concentration the lower the drug release rate [15-17]. The in vitro drug release studies were performed using 900ml of phosphate buffer pH 6.4 as it was not showing good release profile when used pH 7.4 with surfactant 0.5% w/v sodium lauryl sulphate. No significant difference in re-lease percentage was found between lipospheres prepared with 0.05%, 0.075% and 0.1% PVA. The release of OFX loaded lipospheres showed a biphasic profile. The release of OFX from lipospheres showed an initial burst release, which was embedded on the surface of the lipospheres, followed by a slow and continuous release which corresponds to release of OFX entrapped in the lipospheres. The initial burst effect may be attributed as a desired effect to ensure initial thera-

Fig. (3). Effect of surfactant at different concentrations on lipospheres characteristics.

Table 4. Effect of Stirring Speed on Ofloxacin Lipospheres Characteristics (n =3; SD within Approximately 2%).

Batch Stirring Speed (rpm) Mean Diameter ( m) ± SD Yield (%)± SD Entrapment Efficiency (%) ± SD

P 2-1 250 152±1.56 45±0.98 41±1.88

P 2-2 500 137±1.32 47±0.82 43±1.25

P 2-3 750 109±1.02 51±01.02 59±1.01

P 2-4 1000 94±1.78 68±1.35 72±1.27

P 3-1 250 88±1.54 67±0.97 58±0.58

P 3-2 500 85±1.09 68±0.58 70±1.52

P 3-3 750 68±1.76 77±0.59 75±0.49

P 3-4 1000 51±1.92 78±1.41 81±0.74

P 4-1 250 87±1.31 67±0.49 70±0.63

P 4-2 500 81±0.47 72±1.48 76±0.04

P 4-3 750 63±0.96 77±1.27 80±0.92

P 4-4 1000 48±1.02 88±1.07 85±0.83

Note: The batch P4-1, P4-2, P4-3 and P4-4 produced the satisfactory results in terms of yield, particle size and entrapment efficiency. Therefore the tableting characteristics were

carried out only for above mentioned batches.


peutic plasma concentrations of drug. The (Batch P4-1, P4-2, P4-3 and P4-4) in vitro drug release was found in the range of 79.99 % to 98.59 % after the period of 16 hours.

The OFX tableted microspheres were prepared using the excipients as shown in Table 2. The OFX lipospheres were tableted using Avicel PH301 as a directly compressible dilu-ent. The prepared tablets were subjected to various evalua-tion parameters, such as thickness, hardness and drug re-lease. The results of these tests are given in Table 5. Batch TP4-1 and TP4-2 prepared with PVP in the concentration of 1 and 2% w/w, showed lower average hardness (3.4 ± 0.8 kg/cm2). Hence in order to impart sufficient hardness re-quired for the preparation of coated tablets, Batches TP4-3 and TP4-4 were prepared by containing 3 %w/w and 4% w/w of PVP, the hardness of these batches were found to be 4.8 ± 0.6 and 5.6 ± 0.8 kg/cm2 respectively. With the in-crease in hardness, the disintegration time increases (182± 2 s

and 274± 2 s) and the % friability decreases. As batch TP4-4 showed hardness sufficient for maintaining tablet integrity.

In-Vitro Evaluation of OFX Tableted Lipospheres

The dissolution profile of OFX lipospheres (Batch P4-1, P4-2, P4-3 and P4-4) and tableted OFX lipospheres (Batch TP4-1, TP4-2, TP4-3 and TP4-4) was found to be much similar (Fig. 10). The fact of similarity between the dissolu-tion profiles of OFX lipospheres and tableted lipospheres demonstrated that the tableted lipospheres may remain intact after the compression of the OFX lipospheres. If the lipo-spheres would have been broken, the dissolution values would have increased. The amount of drug released at the end of 16th hour from tableted lipospheres was found to be 73.99 to 94.95 %, which reveal that the OFX lipospheres remained intact even after the compression. The drug release rate of lipospheres and tableted lipospheres were not signifi-cantly changed.

Fig. (4). Effect of stirring speed on the particle size of OFX loaded lipospheres.

Fig. (5). Effect of stirring speed on the yield (%) and drug entrapment (%) of OFX loaded lipospheres.


(Fig. 6) Contd….

Fig. (6). The FTIR graph of ofloxacin, cetyl alcohol, PVA and physical mixture respectively.


(Fig. 7) Contd….

Fig. (7). XRD diffractogram of cetyl alcohol, ofloxacin and OFX loaded lipospheres respectively. Table 5. Micromeretic and Tabelting Characteristics of OFX Lipospheres.

Batch Code TP4-1 TP4-2 TP4-3 TP4-4

Bulk density (g mL–1) 0.4±0.24 0.40±0.24 0.4±0.24 0.4±0.24

Tapped density 0.48±0.15 0.49±0.15 0.51±0.15 0.53±0.15

Carr’s index (%) 16.66±0.43 18.36±0.47 21.56±0.43 24.52±0.43

Hausner’s ratio± S.D 1.15±0.19 1.2±0.21 1.2±0.12 1.32±0.17

Micromeretic

evaluation

Angle of repose (°) 24.48±0.21 24.08±0.21 22.97±0.41 22.78±0.22

Average weight (mg) ± SD 271.13±0.21 271.14±0.83 270. 08±0.72 270.03±0.58

Friability (%) 0.50±0.62 0.50±0.42 0.49±0.04 0.48±0.08

Hardness (kg/cm2) ± SD 3.4±0.8 3.4±0.1 4.8±0.6 5.6±0.8

Disintegration (sec) 82± 5 s 88± 4 s 182± 2 s 274±2 s

Tablet evaluation

Parameters

in-vitro drug release (at 16hrs) 73±1.09% 82±0.96% 85±1.24% 94±1.41%

Fig. (8). In vitro release of OFX loaded LS (batch P4-1 to P4-4) in pH 6.4 phosphate buffer solution with 0.5%w/v sodium lauryl sulphate. Each point represents the mean ± SD (n = 3).

Fig. (9). In- vitro release profile of tableted OFX liposphere (batch TP4-1, TP4-2, TP4-3 and TP4-4). Each point represents the mean ± SD (n = 3).


Fig. (10). Comparison of in vitro drug release between lipospheres (P4 -1, P4-2, P4-3 and P4-4) and tableted lipospheres (TP4 -1, TP4-2, TP4-3 and TP4-4). CONCLUSION

Lipospheres containing OFX were successfully prepared by the melt dispersion technique. The melt dispersion tech-nique is very rapid, viable, nontoxic and economical which does not imply the use of organic solvents. The process pa-rameters like surfactant and stirring speed had a major influ-ence on the morphology, entrapment efficiency of the lipo-spheres and drug release which can be optimized to obtain the desirable properties built in the lipospheres. The in vitro drug release study reflected that the improvement of drug solubility in targeted site (lymphatic system) pH 6.4 when compared with same study in pH 7.4. In-vitro release charac-teristics of drug from the microspheres are subject to confir-mation in animal and human studies for coming into conclu-sion of enhanced bioavailability and reduced dose frequency to improve patient compliance.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the Management, Karpagam University, Coimbatore, India and Principal, Kar-pagam College of Pharmacy, Coimbatore, India, Manage-ment, Lincoln University College, Kuala Lumpur, Malaysia, for moral encouragement and providing institutional facili-ties.

PATIENT CONSENT

Declared none.

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Received: June 10, 2012 Revised: July 07, 2012 Accepted: December 20, 2012

2 EFFECT OF PROCEESING VARIABLES OFX LS

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