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Abstract
Liposomes are closed vesicles consisting of one or more concentric spheres of lipid bilayers
(or lamellae) enclosing an equal number of aqueous compartments. Depending on the lipid composition, methods of preparation, and the nature of the encapsulating agents, many types of liposomal products can be formulated. Rational liposome design can be done by selecting appropriate composition and geometry.
Liposomes are characterized in terms of their size and number of lipid bilayers surrounding their central aqueous compartment. Liposomes are broadly classified on the basis of composition or on the basis of their size. They are classified on the basis of their size into:
(1) Multilamellar vesicles (MLV) are distinguished by being larger than 0.1 m in diameter and having more than one bilayer enclosing the aqueous core. They have moderate aqueous volume to lipid ratio (1-4 L/mole lipid).
(2) Large unilamellar vesicles (LUVs) are distinguished by being larger than 0.1 m in diameter and having single bilayer enclosing one aqueous compartment. They have high aqueous volume to lipid ratio (7L/mole lipid).
(3) Small unilamellar vesicles (SUVs) consist of one lipid bilayer compartment and measure around 100 nm or less. They have low aqueous volume to lipid ratio (0.2-1.5 L/mole lipid). SUVs are thermodynamically unstable and susceptible for fusion. Liposomes of different sizes and characteristics usually require different methods of preparation.
The potential advantages that liposomes offer as drug delivery vehicles are clear. They are formulated from naturally occurring membrane components and therefore are biocompatible and biodegradable. They can incorporate both hyDROPhobic and hyDROPhilic drugs as well as biological response modifiers within their structure. Their surface can be modified to alter their biodistribution and pharmacokinetics. Liposomes that are stable in vivo can potentially act as slow-release drug depot.
The discovery of liposomes has opened new opportunities for novel applications in ocular drug delivery, their efficacy as vectors and their ability to increase ocular absorption have ensured their success.
Liposomes offer advantages over most ophthalmic delivery systems in being completely biodegradable and relatively non-toxic. Another potential advantage of liposomes is their ability to come in an intimate contact with the corneal and conjunctival surfaces, thereby, increasing the probability of ocular drug absorption.
Ocular fungal infections, or ophthalmic mycosis, are being increasingly recognized as an important cause of morbidity and blindness. Fungal keratitis is a sight-threatening affliction that requires immediate laboratory diagnosis and prompt therapeutic intervention to prevent loss of the eye.
Fluconazole has a low protein-binding property, and has a predominantly renal excretion. Fluconazole is effectively distributed throughout all tissues, including a high penetration into cerebrospinal fluid, and has also been shown to be non-mutagenic and less toxic than the other azoles.
Fluconazole, by virtue of its pharmacological properties, offers a fresh opportunity for the topical treatment of keratomycosis. The drug acts against all pathogenic Candida species except C. krusei and hence encompasses the majority of fungi which are notorious as causative agents of keratomycosis.
The aim of this thesis was directed to design and prepare fluconazole loaded liposomes. This is a step forward for targeting the release of fluconazole into the cornea of the eye. The prepared liposomes were evaluated for their potential topical use for treatment of Candida keratitis with focusing on their biological efficacy as well as their ability to release fluconazole as antifungal drug.
Accordingly, the work in this thesis considered the following aspects:
1- Preparation and characterization of fluconazole liposomes.
This part deals with the preparation and characterization of fluconazole liposomes prepared by reverse-phase evaporation technique, which was pioneered by Szoka and Papahadjopoulos. The factors influencing the encapsulation of fluconazole into liposomes were investigated to determine the optimum conditions for maximum drug entrapment efficiencies. Amounts of drug, cholesterol, positive charge inducer, negative charge inducer, were added to investigate their effect on fluconazole entrapment efficiency into liposomes and to study their effect on the physical properties of liposomes.
The work in this part comprises:
1- Fluconazole liposomes were prepared using reverse-phase evaporation method. The lipid components (phosphatidylcholine mixed with other lipids such as cholesterol and charge inducing agents) were weighed into 250 ml round bottom flask and dissolved in chloroform. The organic solvent system was slowly evaporated under reduced pressure, using rotary evaporator, at 40oC, such that a thin film of dry lipid was formed on the inner wall of the rotating flask. The lipid film was redissolved in 10 ml ether, and the drug solution in 10 ml acetone together with 5 ml distilled water was added. The mixture was then placed on the rotary evaporator and the organic solvent was removed under reduced pressure.
The liposomes were allowed to equilibrate at room temperature. The liposomal suspension was kept in the refrigerator to mature over night.
The liposomes containing fluconazole were separated from non-entrapped fluconazole by centrifugation a certain volume of the liposomal dispersion at 25000 rpm for one hour at 4oC. The lipo¬somes were separated from the supernatant and one milliliter of the supernatant was diluted and adjusted to volume with methanol in a 10-ml volumetric flask, and the amount of drug was determined spectrophotometrically at λ 261nm.
The percent encapsulation was determined relative to the original drug amount added.
2- The factors influencing the encapsulation of fluconazole into liposomes prepared by reverse-phase evaporation method were investigated to find out the optimal conditions for its entrapment:
a- Effect of drug content using weight ratio of phosphatidaylcholine: cholesterol of 7:4 while increasing the amount of fluconazole added.
b- The effect of increasing cholesterol content, on drug entrapment in liposomes was investigated for liposomal formulae composed of phosphatidylcholine: cholesterol weight ratios of 9:1, 7:2, 7:3, 7:4, 6:4, 7:6 and 5:5 with constant fluconazole content.
c- The effect of adding charge inducing agents either positive or negative, on drug entrapment in liposomes was investigated for liposomal formulae composed of phosphatidylcholine: cholesterol weight ratio 5:5 with constant fluconazole content.
3- Photomicroscopic analysis:
Samples of fluconazole liposomal preparations were examined microscopically at magnification of 1000X.
4-Laser light scattering measurement:
A small aliquot of freshly prepared liposome dispersion sample was used to characterize the particle size and size distribution, by light scattering based on laser diffraction technique. The particle size analysis was carried out on freshly prepared neutral, positively and negatively charged liposomal dispersions of different weight ratios.
5- Differential Scanning Calorimetry (DSC) Measurements:
Differential Scanning Calorimetry (DSC) experiments were performed with differential scanning calorimeter. Samples of fluconazole, cholesterol, empty and drug loaded liposomes composed of PC: Ch (7:3 or 5:5) weight ratios were submitted to DSC analysis.
The results of this work revealed the following:
1- Increasing the amount of added fluconazole during the formulation from 5 mg to 25 mg significantly increased the percent of drug encapsulation by the liposomes.
2- Drug encapsulation in different weight ratios of phosphotidylcholine: cholesterol of 9:1, 7:2, 7:3, 7:4, 6:4, 7:6 and 5:5 were evaluated showing that, the encapsulation percent of fluconazole significantly increased as the amount of cholesterol increased up to 30% at the ratio (PC:Ch; 7:3). Further increase in the amount of cholesterol produced a decrease in the encapsulation percent, especially at the ratio (PC: Ch; 7:6).
3- Incorporation of the positively charged stearylamine into liposomes composed of different weight ratios of phosphatidylcholine and cholesterol resulted in a significant decrease of the entrapment percent of fluconazole into liposomes at ratio (PC: Ch: SA; 5:5:0.25) followed by a slight increase in the entrapment of fluconazole into liposomes relative to the encapsulation in neutral liposomes.
However, incorporation of the negatively charged dicetyl phosphate into liposomes resulted in a significant increase in the amount of fluconazole entrapped in the liposomes in comparison with the neutral liposomes at all tested ratios.
4- Liposomes prepared by reverse-phase evaporation method reveal the presence of homogeneous population of unilamellar vesicles with one phospholipids bilayer and oligolamellar vesicles consisting of a few concentric bilayers.
5- The particle size frequency distribution of neutral liposomes composed of phosphatidylcholine: cholesterol in the different weight ratios revealed that, incorporation of more cholesterol would yield significantly larger vesicles, while that of positively-charged liposomes resulted in a significant decrease in particle size frequency distribution.
Moreover, the particle size frequency distribution of negatively charged liposomes showed an insignificant decrease in the particle size.
6- DSC thermograms of empty aqueous liposomal dispersion showed that, all the lipid components interact with each other to a great extent while forming the lipid bilayer. DSC thermograms of the fluconazole-loaded liposomal dispersion showed that, the incorporated fluconazole, associated with the lipid bilayers, interacted to a large extent with them, and perturbed them. Absence of the melting endotherm of fluconazole suggested significant interaction of drug with bilayer structure.
2- In-vitro release of fluconazole from liposomes:
In order to study the drug release from liposomes with different compositions, dialysis method was applied using Spectra/ Por® dialysis membrane of 12000-14000 molecular weight cut off. The release media was Sّrensen`s modified phosphate buffer (pH=7).
The results of this work revealed the following:
1- Liposomes showed a slow release rate of the drug in comparison to fluconazole solution.
2- There was no significant difference in the release extent of fluconazole from liposomes containing different amounts of the drug.
3- Increasing cholesterol weight ratio in the prepared liposomal formulations progressively decreased the release of fluconazole from the vesicles. Further increase in the amount of cholesterol in the formula (PC: Ch; 5: 5), resulted in a significant increase in the release of drug.
4- It is clear that, the positively charged liposomes showed slower rate of drug release compared to neutral ones.
5- Negatively charged liposomes showed slight increase in the release rate and extent of fluconazole after 6 hours for the liposomal formulations 5:5:0.25 and 5:5:0.5 in comparison with neutral. Further increase in the amount of dicetyl phosphate 5:5:1 resulted in a significant decrease in the release rate.
6- Release pattern of fluconazole from liposomes followed diffusion controlled or first order mechanism.
3- In-vitro antifungal activity of fluconazole loaded liposomes:
This was performed by disc diffusion method; sterile filter papers, were impregnated with 20 µl of different concentrations of fluconazole solutions and fluconazole loaded liposomes and placed on the surface of the inoculated sabouraud’s dextrose agar in triplicate, for each tested strains. Discs without antifungal agents were placed on inoculated agar for positive growth control results. All inoculated plates where incubated at 30°C for 48 hours and examined for the inhibition zone. The diameter of the inhibition zone around each disc was measured and the mean value of three experiments was calculated.
The results showed that,
1- All tested fluconazole concentrations produce good inhibition effect with Candida species while the Fusarium species (F. Verbicilloides and F. Solani) and Aspergillus species (A. Flavus and A. Fumigatus) were considered as resistant strains.
2- No significant difference between all liposomal formulae and fluconazole solution with good inhibition effect except with the liposomal formulae PC: Ch (7:4 and 6:4) as well as PC: Ch: SA (5: 5: 0.5) there was a little significant difference. Fluconazole free liposomes (control) had no antifungal activity.
3- Increasing cholesterol amount led to significant decrease in the diameter of growth inhibition zone.
4- The effect of stearylamine (SA) as a positive charge inducer on the antifungal activity of fluconazole loaded liposomes resulted in a decrease in the mean diameter of inhibition zone, especially at the liposomal formulae 5:5:0.5 although it is statistically insignificant compared to neutral liposomes.
5- The addition of dicetyl phosphate (DP) resulted in apparent increase in the mean diameter of inhibition zone, at all tested ratios although it is statistically insignificant compared to neutral liposomes.
from the above results, the liposomal formulae composed of PC: Ch weight ratios of 7:4 as neutral liposomes could be used in further investigations, for its high half-life (t1/2).
Neutral liposomes PC: Ch 5:5 was also used for the following investigations for high release rate and also as comparison for the other liposomal formulae composed of (PC: Ch: SA weight ratio of 5:5:0.5) as positively charged liposomes and (PC: Ch: DP weight ratio of 5:5:1) as negatively charged liposomes which were also used in the following investigations, for their high half-life (t1/2).
4- Stability studies of fluconazole liposomes:
Stability of liposomes is an important factor in their development, evaluation and confidence in application. Physical stability study of fluconazole liposomes was carried out to determine the comparative leakage of the drug from liposomes (in a liquid form) stored at different condition compared to each other. The above-mentioned four liposomal formulae of choice were investigated.
The work done in this part includes:
The optimized fluconazole liposomal eye DROPs formulations were stored in tightly sealed 20 ml glass vials in refrigerator at temperature (5°C) and at room temperature (25˚C) for 24 weeks.
The pH values and the viscosity were measured at both temperatures directly after preparation and at the end of the storage period (24 weeks).
Samples of liposomal dispersions were regularly tested at time intervals of 0, 4,8,12 and 24 weeks for the following attributes at each temperature:
(i) Signs of sedimentation or creaming if any, and change in color of the dispersions.
(ii) The formulae were analyzed using laser diffraction particle size analyzer (Mastersizer X).
(iii) Extent of leakage:
At predetermined time intervals of 2, 4,6,8,10,12 and 24 weeks, an aliquot (1ml) of the stored samples of each formulation was centrifuged in a cooling centrifuge at 25000 rpm for one hour to separate the free drug leaked out of the liposome during the storage period. The amount of free drug in the supernatant was measured spectrophotometrically at λ 261nm.
The results of this part revealed the following:
1- Liposomes can be arranged in descending order according to their stability as follow, negatively charged (PC:Ch:DP; 5:5:1) > positively charged liposomes (PC:Ch:SA; 5:50.5) > neutral liposomes (PC:Ch; 5:5) > neutral liposomes (PC:Ch; 7:4).
2- Negatively charged liposomes (PC: Ch: DP; 5:5:1) was found to be reasonably stable in terms of aggregation, fusion and/or vesicle disruption tendencies, over the studied storage period either in refrigerator or at room temperature.
3- Negatively charged liposomes show better stability, manifested in higher drug retention followed by the positively-charged liposomes then the neutral ones.
4- Maximum stability was observed for the formulae stored at temperature (5˚C) wherein the liposomes retained their normal structure and size and minimum leakage of the active ingredients.
5- In- vivo antifungal evaluation (Experimental Candida keratitis in rabbits):
In the present study, a well-established rabbit model of fungal keratitis was used to investigate the potential of fluconazole in the therapy of deep keratitis due to C. albicans. The model is reproducible without the need of immunosuppressive pretreatment. Therapy is started only on day 2, when stromal keratitis is manifested, hence the model was more closely parallels to human corneal candidiasis.
The rabbits were sedated by the intraperitoneal injection of 0.5 ml Thiopental®. Intrastromal injection of 10μl of inoculum (Candida albicans, containing 2.5 x 105 cell), was done in both eyes by inserting a sterile 27-gauge needle into the central corneal stroma tangential to the corneal surface to a depth of one half of the corneal thickness.
This study included forty rabbits, were randomly divided into five equal groups:
• The first group (8 rabbits), the right eyes received fluconazole solution (0.2%w/w).
• Second group (8 rabbits), the right eyes received neutral fluconazole loaded liposomes (PC: Ch; 5:5) (0.2%w/w).
• Third group (8 rabbits), the right eyes received neutral fluconazole liposomes (PC: Ch; 7: 4) (0.2%w/w).
• Fourth group (8 rabbits), the right eyes received negatively charged fluconazole liposomes (PC: Ch: DP; 5:5:1) (0.2%w/w).
• Fifth group (8 rabbits), the right eyes received positively charged fluconazole liposome (PC: Ch: SA; 5: 5:0.5) (0.2%w/w).
The left eyes of each rabbit did not get any treatment and considered as control.
Eye DROPs were instilled into the conjunctival sac of the rabbits after 48 hours of the inoculation procedure. The instillation continues every 3 hours for 12 hours (four times daily) in the first three days, then every four hours (three times daily) in the next period of treatment. After instillation the rabbit eye is closed for approximately two minutes. The rabbits eyes were examined daily over a 21-day period by hand- held torch for signs of infection, and the severity of inflammatory reaction was noted (hypopyons, iritis) by the ophthalmologist. Photographs were taken after the induction of keratitis and before treatments, during treatment period and at the end of treatments in order to find out signs of improvement.
The results of this work revealed the following:
1- Rabbits infected with C. albicans responded better and showed improvement in size of ulcer and hypopyons improved on using fluconazole-loaded liposomal formulae than fluconazole in solution.
2- Comparing rabbits’ corneas, treated with different liposomal formulations, group 4 which received fluconazole loaded negative liposomes PC: Ch: DP 5:5:1 showed better improvement than positive and neutral formulations.
3- Signs of corneal healing were observed in all groups treated with fluconazole in solution or liposomal formulation with variation in percent of healing and time of reaching healing.
4- The groups are arranged according to the time to reach complete healing as: group 4< group5 < group 3< group 2< group 1.
6- Clinical studies on human patients having Candida keratitis:
The negative liposomal formula (PC:Ch:DP; 5:5:1) loaded with fluconazole which showed high activity against infected rabbit’s eye was chosen for the clinical study.
Five patients, four males and one female having Candida keratitis with indolent ulcers were chosen among patients presented to Department of Ophthalmology, Assiut University Hospitals.
Treating those patients with the prepared fluconazole loaded negative liposomes (0.2%w/w) was done by instructing them to apply the eye DROPs four times daily for the first two weeks of treatment and then three times daily for the end of treatment period..
There was an improvement in size and depth of the ulcer in three patients with appreciable improvement in visual acuity, while the other two patients did not improve due to late presentation of these cases.
It could be concluded that, the findings obtained from in-vitro and in-vivo studies demonstrate the efficacy of liposomes as ocular drug delivery system. Therapy with topical fluconazole liposomes (2 mg/ml) was successful to a great extent and resulted in a fairly high bioavailability and high efficacy in eliminating Candida albicans infection of the cornea (Candida keratitis).