Journal of Liposome Research, 2010; 20(1): 31–37

R E S E A R C H A R T I C L E

Optimization of gatifloxacin liposomal hydrogel for enhanced transcorneal permeation

Khaled Mohamed Hosny1,2

1Department of Pharmaceutics, King Abdulaziz University, Jeddah, Saudi Arabia, and 2Department of Pharmaceutics, Beni Sueif University, Egypt

Address for Correspondence: Khaled Mohamed Hosny, Department of Pharmaceutics, King Abdulaziz University, Jeddah, Saudi Arabia; Fax: +99626951696; E-mail: elswaify2000@yahoo.com

(Received 18 January 2009; revised 5 May 2009; accepted 10 May 2009)

Introduction

Drug delivery in ocular therapy has long been a difficult task. Poor bioavailability of drugs from conventional ocular dosage forms is mainly due to the precorneal loss factors, which include tear dynamics, insufficient resi-dence time in the conjunctival sac, and nonproductive absorption (Le Bourlais et al., 1998; Sultana et al., 2006; Budai et al., 2007).

Fluoroquinolones, as a group, have shown excellent activity against the most frequently occurring gram-pos-itive and -negative ocular pathogens (Cochereau-Massin et al., 1993; Adenis et al., 1995; Diamond et al., 1995; Gross et al., 1997; Jauch et al. 1999). Earlier generation fluoroquinolones, such as ciprofloxacin and ofloxacin, have been used widely to treat various pathogenic

conditions. However, the development of a resistant strain against these fluroquinolone has been reported (Chaudhry et al., 1999; Garg et al., 1999).

Gatifloxacin, a fourth-generation fluoroquinolone, possesses an improved antibacterial spectrum, particu-larly against resistant staphylococcus and streptococcus pathogens, compared with older fluoroquinolones (Perry et al., 2002; Jensen et al., 2005). Efficacy of the marketed ophthalmic fluoroquinolone products, mostly aqueous solutions, is limited by poor ocular bioavailability (Lin et al., 1996; Wiechens et al., 1998–1999), compelling the frequent dosing regimen and the concomitant patient compliance.

In order to improve ophthalmologic bioavailability of drugs and to prolong the therapeutic action, intro-duction of novel delivery systems, including vesicular

ISSN 0898-2104 print/ISSN 1532-2394 online © 2010 Informa UK LtdDOI: 10.3109/08982100903030255

AbstractThe aim of this study was to prepare and characterize a topically effective prolonged-release ophthalmic gatifloxacin liposomal hydrogel formulation. Reverse-phase evaporation was used for the preparation of liposomes consisting of phosphatidylcholine (PC) and cholesterol (CH). The effect of PC:CH molar ratio on the percentage of drug encapsulated was investigated. The effect of additives, such as stearylamine (SA) or dicetyl phosphate (DP), as positive and negative charge inducers, respectively, was studied. Morphology, mean size, encapsulation efficiency, and in vitro release of gatifloxacin from liposomes were evaluated. For hydrogel preparation, carbopol 940 was applied. In vitro transcorneal permeation through excised albino rabbit cornea was also determined. Optimal encapsulation efficiency was found at the 5:3 PC:CH molar ratio; by increasing CH content above this limit, the encapsulation efficiency decreased. Positively charged liposomes showed superior entrapment efficiency over other liposomes. Hydrogel-containing liposomes with lipid content PC, CH, and SA in a molar ratio of 5:3:1, respectively, showed best release and transcorneal permeation. These results suggest that the encapsulation of gatifloxacin into liposomes prolonged the in vitro release, depending on composition of the vesicles. In addition, the polymer hydrogel used in the preparation ensured steady, prolonged transcorneal permeation. In conclusion, gatifloxacin liposomal hydrogel is a suitable delivery system for the improvement of the ocular bioavailability of gatifloxacin.

Keywords: Gatifloxacin; liposome; ocular drug delivery system; transcorneal permeation; hydrogel

http://www.informahealthcare.com/lpr

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drug-delivery systems, such as liposomes, have recently been proposed (Bangham et al., 1965; Gregoriadis and Florence, 1993; Farkas et al., 2004; Kaur et al., 2004).

Liposomes have been investigated since 1970 as a system for the delivery or targeting of drugs to specific sites in the body. Some liposomal drug-delivery systems exhibited superior pharmacological properties to those observed with the conventional formulation (Foradada and Estelrich, 1995). Activity of liposomes as a carrier for drugs depends upon various factors, such as charge, rigidity, composition of the liposomal membrane, encap-sulation efficiency, and release rate (Choudhari et al., 1994; Galovic Rengel et al., 2002; Karathanasis et al., 2005; Nii and Ishii, 2005). Liposomes can be formulated from a variety of lipid and lipid mixtures with different com-position (Aranda et al., 2005), and they can be modified in particle size, structure, and surface charge to obtain desirable physicochemical properties to suit particular needs (Law and Hung, 1998; El-Samaligy et al., 2006).

Recently, due to the low viscosity of colloidal suspen-sions of liposomes that does not allow sufficient reten-tion time of the dosage form at the site of action, different hydrogel matrices, such as carbopol 940, have been used to increase the viscosity of the topical preparation and to increase the retention time of the formulation at the site of administration due to good bioadhesive properties (Durrani et al., 1992; Budai et al., 2007). In addition, the liposomal hydrogel ensures a steady, prolonged release of gatifloxacin from liposomes and makes intimate contact with the corneal and conjunctival surfaces. The aim of the present study was to prepare and character-ize a topically effective, prolonged-release ophthalmic gatifloxacin liposomal-based hydrogel formulation. The factors influencing the encapsulation of gatifloxacin into liposomes were investigated, and a characteriza-tion of the prepared liposomes regarding morphology, particle size, and in vitro drug release was performed, liposomal-based hydrogel was prepared, and an in vitro transcorneal permeation study was carried out to inves-tigate the permeation properties of gatifloxacin from the optimized liposomal hydrogel formulation.

Materials and methods

Materials

Gatifloxacin, phosphatidylcholine from egg yolk, and cholesterol were purchased from Sigma Chemical Company (St. Louis, Missouri, USA). Stearylamine and dicetyl phosphate were obtained from Fluka Chemical Company (Buchs, Germany). Carbopol 940 was obtained fromBF Goodrich Company (Saginaw, Michigan, USA). Triethanolamine was obtained from Junsei Chemical Company (Tokyo, Japan). All other reagents were

commercially available and of analytical grade. Spectra/Pore® dialysis membrane (12,000–14,000 molecular-weight cut-off) was purchased from Spectrum Laboratories Inc. (Broadwich st., Rancho Dominguez, USA).

Methods

Preparation of liposomesGatifloxacin liposomes were prepared by using the reverse-phase evaporation technique (Szoka and Papahadjopoulos, 1978). The lipid component (300 mg), including PC and CH, either alone or mixed with SA or DP, were accurately weighed into a round-bottomed flask and dissolved in a chloroform:methanol mixture (1:2 v/v). A thin lipid film was formed on the inner side of the flask by evaporating the organic solvents under a vacuum, using a rotary evaporator at 40°C (Buchi R-110 Rotavapor; Postfach, Switzerland). The lipid film was redissolved in 10 mL of ether, and the drug solu-tion (30 mg), in 10 mL of acetone together with 10 mL of phosphate-buffered saline (PBS; pH 7.4), was added. The mixture was then placed on the rotary evaporator to evaporate the organic solvent. The liposomes were allowed to equilibrate at room temperature, and 10 mL of PBS was added to the liposomal suspension, which was kept in the refrigerator overnight. Finally, the liposomal suspension was filtered through a 0.22-µm microporous filter and kept in a refrigerator.

Determination of gatifloxacin entrapment efficiency in liposomesTo determine the encapsulation efficiency of lipo-somes, a known amount of liposomes was centrifuged at 100,000 rpm for 30 minutes at 4°C. The liposomes were separated from the supernatant and sonicated with methanol for 15 minutes. The concentration of gatifloxacin in methanol was determined spectrophoto-metrically at 293 nm in a spectrophotometer (UV-1601 PC; Shimadzu, Kyoto, Japan). The entrapment efficiency (EE%) was calculated as shown in Equation 1:

EE [Me Mt] 100% = / × % (1)

where Me and Mt represent the encapsulated gatifloxacin and total gatifloxacin amount, respectively. EE% was determined to evaluate the effect of PC:CH molar ratios on EE% by using different molar ratios for PC:CH ( 5:1, 5:2, 5:3, 5:4, and 5:5) with constant added gatifloxacin content. In addition, EE% was determined to study effect of adding a positive (SA) and negative charge inducer (DP) on EE% of gatifloxacin in liposomes.

Morphology and size analysis of liposomesThe physical morphology of samples of gatifloxacin liposomes composed of PC:CH in the molar ratio of 5:3

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Optimization of gatifloxacin liposomal hydrogel 33

were examined by using a transmission electron micro-scope (EM 10; Zeiss, Oberkochen, Germany). A drop of liposome dispersion sample was applied on a 300-mesh collodion copper grid on paraffin and left for 10 minutes to allow some of the liposome powder to adhere on the collodion. The remaining dispersion was removed, and a drop of 2% aqueous solution of uranyl acetate was applied for 2 minutes. The remaining solution was then removed and the sample was air-dried, prior to measurement with a transmission electron microscopeThe mean particle size was determined by using laser diffraction particle size analysis (Malvern Instruments Ltd., Malvern, UK).

In vitro drug-release studiesThe in vitro release of gatifloxacin from neutral, posi-tively, and negatively charged liposomes was measured by using the dialysis method. The dialysis method was applied by using a Spectra/Pore dialysis membrane (12,000–14,000 molecular-weight cut-off). This mem-brane assures the permeation of the drug at same time it retains liposomal forms.

An accurately measured amount of gatifloxacin lipo-somal suspension, equivalent to 3 mg of gatifloxacin, was suspended in 1 mL of PBS (pH 7.4) in a glass cylin-der with a length of 8 cm and a diameter of 2. 5 cm. This cylinder was fitted, before the addition of the liposomal suspension, with a presoaked dialysis membrane and was suspended in the dissolution flask of the United States Pharmacopoeia (USP) dissolution tester (Pharma Test, Hainburg, Germany) containing 75 mL of PBS (pH 7.4) and maintained at a temperature of 37°C. The glass cylinder was adjusted to rotate at a constant speed (75 rpm). At predetermined time intervals (0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours), 4 mL of the release medium were withdrawn and assayed spectrophotometrically for drug content at 293 nm.

Preparation of gatifloxacin liposomal hydrogelHydrogel dosage form of gatifloxacin liposomes were prepared by using deionized water as a vehicle and car-bopol 940 as a gelling agent at a concentration of 1.5% (w/w). After complete hydration of the carbopol by the vehicle, an accurate amount of gatifloxacin liposomes, composed of PC, Ch, and SA in a molar ratio (5:3:1), were added to prepare the hydrogel with a concentration of 0.3% (w/w) of the drug. Then, triethanolamine was added and mixed completely until the gel was formed.

In vitro drug transcorneal permeationThe transcorneal experiment was performed with a conventional diffusion chamber. Albino rabbits were humanely killed by an intravenous injection of excess sodium phenobarbital, and the whole eyes were enu-cleated. The method of dissection of the cornea was the same as those described previously (Malhotra

et al., 1997). The corneas of both eyes were excised and then mounted on the diffusion chamber. The receptor compartment was filled with 10 mL of PBS (pH 7.4), while 1 g of hydrogel containing gatifloxacin liposomes (neutral, negative, and positive charged liposomes), or 1 g of gatifloxacin hydrogel (composed of pure drug dispersed in hydrogel), or 1 mL of gatifloxacin solution (composed of pure drug dispersed in distilled water) were applied to the epithelial side. The temperature in the diffusion chamber was maintained at 34°C by ther-mostatic water bath. The receptor buffer was removed 0.5, 1, 1.5, 2, 3, 4, and 6 hours and immediately replaced by previously aerated fresh receptor buffer. The samples were analyzed spectrophotometrically for drug content at 293 nm. Results were expressed as amount permeated and percent permeation or in vitro ocular availability, as shown in Equation 2:

Permeation ( ) [Ap At] 100% = / × (2)

where Ap and At represent the amount of gatifloxacin permeated in the receptor and the initial amount of gatifloxacin in the donor, respectively.

Results and discussion

Factors affecting entrapment efficiency

Figure 1 shows the effect of a molar ratio of PC:CH on the EE% of gatifloxacin in liposomes. As observed, the EE% increased by increasing the CH content until a certain limit was reached, above which the EE% decrease by increasing the CH concentration. The maximum EE% of gatifloxacin into liposomes (85.74 ± 4.03) was obtained from liposomes with a PC:CH molar ratio (5:3). The one-way analysis of variance (ANOVA) showed that there was a significant difference in EE% between liposomes with different molar ratios of PC:CH (P < 0.001). The increase in EE% by increased CH content occured because by

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EE%

Figure 1. Effect of molar ratio of PC:CH on gatifloxacin entrapment efficiency (EE%) (mean ± SD; n = 3).

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increasing the cholesterol concentration in the lipid bilayer, the rigidity of the bilayer increased, resulting in higher stability and reduced permeability of the lipo-somal membrane (du Plessis et al., 1996) and, hence, greater drug retention (Perugini and Pavanetto, 1998). Decreasing the EE% with an increasing cholesterol ratio above a certain limit may be due to the fact that increas-ing cholesterol above a certain concentration can disrupt the regular linear structure of a liposomal membrane (New, 1990). Thus, the ratio of 5:3 (PC:CH) was the most efficient regarding EE%.

The effect of charge-inducing agents on the EE% of gatifloxacin in liposomes containing PC:CH in the molar ratio of 5:3 is shown in Figure 2. The positively charged liposomes containing PC:CH:SA in the molar ratio of 5:3:1exhibited the highest EE% (92.23 ± 1.72), followed by neutral ones containing PC:CH in the molar ratio of 5:3 (85.74 ± 4.03), and then by negatively charged ones containing PC:CH:DP in the molar ratio of 5:3:1 (77.12 ± 2.74). This order of EE% would result because gatifloxacin has a negatively charge carboxylic group, so an electrostatic attraction would occur between the drug and the positively charged SA, with this attraction leading to increasing EE%, when compared with neutral liposomes. On the other hand, electrostatic repulsion would occur between the drug and negatively charged DP, lead to a decrease in EE% less than that for neutral liposomes. Similar results were also obtained during the incorporation of charge-inducing agents into aza-thioprine liposomes (Gulati et al., 1998), indomethacin liposomes (Srinath et al., 2000), and acetazolamide lipo-somes (Rania et al., 2007).

Morphology and size analysis of liposomes

Figure 3 shows the gatifloxacin liposomes prepared by the reverse-phase evaporation method by a molar ratio of 5:3 (PC:CH), revealing the presence of homogenous unilamellar vesicles with one phospholipid bilayer. As observed, they were well-identified spheres with a large internal aqueous space, and they exist in dispersed and aggregate collections.

The results of particle-size analysis were 1,960, 1,840, and 1,750 nm for positive, negative, and neutral lipo-somes, respectively. This order would result because in positively charged liposomes, many factors lead to increased size: first, the inclusion of charge leads to increased spacing between the adjacent bilayer (Nagarsenker et al., 1999); second, the inclusion of a large amount of gatifloxacin, as mentioned in EE% determination; third, the positive charge electrostati-cally attracted the drug, which pushed the phospholip-ids head group apart, leading to increasing the particle diameters (Gruner, 1997). The particle diameter of nega-tively charged liposomes was also larger than the neutral

ones, which was due to the fact that inclusion of charge leads to increased spacing between adjacent bilayers.

In vitro drug release study

Figure 4 shows the release profile of gatifloxacin from neutral, positive, and negatively charged liposomes. From all release profiles, it was obvious that the nega-tively charged liposomes showed the highest rate and extent of drug release, followed by neutral, and, finally, the positively charged ones, which gave more prolonged, controlled release. This order may be due to electrostatic attraction forces that may exist between the anion drug and amino moiety of SA, while electrostatic repulsion may occur between the drug and negatively charged

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Figure 2. Effect of charge on gatifloxacin entrapment efficiency (EE%) (mean ± SD; n = 3).

Figure 3. Transmission electron microphotograph of gatifloxacin reverse-phase evaporation liposomes composed of PC:CH (5:3) molar ratio. Magnification 10,000X.

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Optimization of gatifloxacin liposomal hydrogel 35

liposomes, resulting in a higher rate of drug release. One-way ANOVA revealed that the difference was not significant (P < 0.01). Due to the nonsignificant differ-ence in the in vitro release, and due also to higher EE% of positively charged liposomes, as mentioned before, a positively charged liposome with lipid content PC, CH, and SA in the molar ratio of 5:3:1 was chosen for the preparation of the liposomal hydrogel, in order to pre-pare a topically effective, prolonged-release ophthalmic gatifloxacin formulation.

In vitro drug transcorneal permeation

Figure 5 shows the transcorneal permeation profile of gatifloxacin from its different formulations. Concerning to the effect of charge on transcorneal permeation profile of gatifloxacin from differently charged 0.3% liposomal hydrogel, it was clear that the cumulative amounts of gatifloxacin permeated after 6 hours were 975, 850, and 716 µg from positively, neutral, and negatively charged liposomes, respectively. This may be explained by the fact that an electrostatic attraction force occured between positively charged liposomes and the negatively charged corneal membrane; this attraction enhanced the transcorneal permeation of gatifloxacin from positively charged liposomes with lipid composition PC:CH:SA in the molar ratio of 5:3:1 more than neutral liposomes. In the case of negatively charged liposomes, the cumu-lative amount was less than that of neutral liposomes; this may be due to an electrostatic repulsion force that occured between negatively charged liposomes and the negatively charge corneal membrane. This repulsion decreased the transcorneal permeation of gatifloxacin from negatively charged liposomes with lipid composi-tion PC:CH:DP, in the molar ratio of 5:3:1, less than that of neutral liposomes.

Figure 5 also shows the transcorneal permeation profile of gatifloxacin from its 0.3% aqueous solution

and 0.3% hydrogel. The extent of drug permeation after 6 hours from liposomal hydrogel and aqueous solution was significantly different. The cumulative amount per-meated from hydrogel containing free gatifloxacin was 515 ± 16.3 µg and the percent permeated was 17.1%. But, the cumulative amount permeated from aqueous solution was 250 ± 9.4 µg only, and the percent perme-ated was 8.3%; this means that gatifloxacin hydrogel showed a 2-fold higher permeation percentage than aqueous solution. This may explained by the fact that the mucoadhesive properties of hydrogel base ensure an intimate contact between the drug and corneal mem-brane, which prolong the retention of the drug at the site of administration, which is beneficial for enhancing per-meation. While positively charged liposomal hydrogel shows a 4-fold higher permeation than aqueous solution, this may be explained by the fact that an electrostatic attraction force occured between positively charged liposomes and the negatively charge corneal mem-brane. We speculate, then, that liposomes, adsorbed to the corneal surface, transfer their membrane-associated drug directly to the corneal epithelial cell membranes, thereby facilitating drug transport across the cornea (Singh and Mezei, 1983; Lee et al., 1985). Other mecha-nisms, such as endocytosis of liposomes or fusion of liposome membrane with the plasmalemma, are also involved in enhanced transport (Schaeffer and Krohn, 1982). In addition, the mucoadhesive properties of the hydrogel base ensure an intimate contact between liposomes and the corneal membrane that prolongs the retention of the formulation at the site of administration, which is beneficial for enhancing permeation (Kaur and Smitha, 2002; Deepika and Indu, 2005).

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Figure 4. Effect of charge on release profile of gatifloxacin from lipo-somes (mean ± SD; n = 3).

Positively Charged Gatifloxacin Liposomal hydrogelNegatively Charged Gatifloxacin Liposomal HydrogelNeutral Gatifloxacin Liposomal HydrogelGatifloxacin HydrogelGatifloxacin Solution

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Figure 5. Transcorneal permeation profile of differently charged gatifloxacin liposomal hydrogel and gatifloxacin hydrogel, in com-parison to its aqueous solution (mean ± SD; n = 3).

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Conclusions

On the basis of these studies, it can be concluded that the formulation of gatifloxacin as liposomal hydrogel will provide the maximum in vitro ocular availability through albino rabbit cornea. Addition of cholesterol in liposomes increases gatifloxacin encapsulation effi-ciency until a certain concentration is reached, above which the encapsulation efficiency decreases by increas-ing the cholesterol concentration. The addition of stear-ylamine as a positive charge inducer enhances encapsu-lation efficiency, while the addition of dicetyl phosphate decreases encapsulation efficiency. Dispersion of lipo-somes in hydrogel matrix ensures a steady, prolonged release and transcorneal permeation of gatifloxacin, which improves ocular bioavailability.

Acknowledgements

Declaration of interest: The authors report no financial conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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