Screening of effective formulation techniques for Designing and Fabrication of Terbinafine hydrochloride ethosomes

 

Ashok A. Hajare*, Hemalata S. Dol

Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy,

Near Chitranagari, Kolhapur - 416103, (M.S.) India.

 

ABSTRACT:

The present investigation aimed to design and fabricate terbinafine HCl (TH) loaded ethosomes for enhancement of transdermal delivery in the management of fungal diseases. The TH loaded ethosomes were explored initially by the selection of appropriate method amongst reported one and further optimized for best amongst the investigated. In present study, TH loaded ethosomes were prepared using cold method, ethanol injection method, and mechanical dispersion using rotary evaporator method and assessed for vesicle size, drug entrapment, zeta potential, and polydispersity index (PDI). The mechanical dispersion using thin film hydration method (vesicle size 126.6±1.55nm, entrapment efficiency 82.10±0.25%) was selected as an appropriate for development of TH ethosomes. Ethosomes prepared using thin film hydration method was optimized on the basis of concentration of TH: PL 90H (1:2, 1:4, 1:6 and, 1:8), hydroethanol 20-45%, volume of ethanol, stirring speed and sonication time. The comprehensive analysis revealed that concentration of PL 90H (TH: PL 90H ratio 1:2, 1:4, 1:6), hydroethanol 20-40% w/v with 10 mL ethanol for film formation following stirring speed 60 rpm, 100 rpm for rehydration of film and 30 min ultra-bath sonication were desired parameters for development of TH ethosomes. The aforementioned optimization results observed in the limit for vesicle size 105.56±2.77nm to 178.71±2.07 nm, entrapment efficiency 63.04±0.67% to 89.52±0.87% with -31 mV to -24 mV zeta potential indicating stability of vesicles. The recorded results indicate that mentioned optimization aspects can be useful for development of drug loaded ethosome with factorial design approach.

 

 

 

INTRODUCTION:

Terbinafine hydrochloride (TH) is a widely prescribed an antifungal antibiotic for the systemic use in the treatment of dermatophytes (Trichophyton, Epidermophyton and Microspora) along with tinea infections¹. Its oral administration is associated with systemic adverse effects due to the longer duration of therapy of about 3 months with dose of a single tablet. Due to these impediments, the transdermal route is a recommended as an alternate route to its oral administration². Chemically TH is highly lipophilic and its transdermal application has potential for site-specific delivery with an improved physiological and pharmacological response.

 

 

 

Transdermal drug delivery (TDD) felt on the short-comings of other prevailing drug delivery. It is a non-invasive delivery that can be exploited to circumvent the limitations of oral drug therapy^3,4. Lipid carrier systems due to biocompatibility with skin lipids offer excellent candidature for transdermal application. Touitou et al. in the form of ethosome have recently developed a novel approach, which is predominantly a lipid carrier^3,5. Ethosomes are tiny soft vesicles that are capable of entrapping either hydrophilic, hydrophobic, or amphiphilic drugs and contain 20 to 45% of alcohol that makes it unique for enhanced transdermal delivery ^6. Ethosomes are predominantly composed of phospholipids and ethanol. The high penetration rate of ethosomes is due to its small vesicular structure and synergistic effect of phospholipid with a high content of ethanol that enables it to access easily through the stratum corneum barrier of the skin^6,7. Further, the research has proved that entrapment of drug in vesicles may help to localize delivery of drug and enhance solubility and availability of drug at the site for systemic action which in turn may reduce dose and systemic side effects^8,9.

 

Touitou et al. addressed hot and cold two methods for the fabrication of ethosomes. Further attempts have been made by researcher to modify these techniques by inclusion of mechanical dispersion using the thin film hydration (TFH), ethanol injection, classical dispersion method etc.¹⁰. The preparation method, processing parameters and concentrations of excipients affect the ethosome formulation for a particular drug. Many research studies reported on ethosomes are either focused on formulation variables impact on characteristics of ethosomes; mechanism of enhanced skin penetration; or comparative assessment of ethosome with either liposome or conventional marketed formulation. These studies have not explored the impact of drug, an excipients and formulation technique for development of ethosome⁹.

 

The present research was aimed to explore the screening of effective formulation method amongst cold, ethanol injection and mechanical dispersion method for fabrication of TH ethosomal system and study its effect on vesicle size (nm), entrapment efficiency (%) and PDI value. In addition, an attempt was made to demonstrate the most effective method for fabrication of TH ethosomes and its processing and formulation parameters. The outcome of this work would be useful to understand the interaction of formulation variables and impact of formulation methodology in-depth for entrapment of TH.

 

MATERIALS AND METHODS:

Phospholipon 90H obtained as a gift sample from Lipoid (Germany), ethanol purchased from Loba Chem Pvt. Ltd. (Mumbai), Cholesterol purchased from Nice Chemicals (Mumbai) and all the other ingredients used were of analytical grade.

 

Preparation of TH ethosomes:

TH ethosomes were prepared by minor modification in the method as reported by Touitou et al. ¹⁰. For the present research study, a cold method using a magnetic stirrer, mechanical dispersion with rotary flash evaporator and ethanol injection method using homogenizer were used^10,11. During development amount of drug and excipients were kept constant for each method and high proficiency processing parameters were selected for primary screening. The vesicle formulation composition was TH (10mg), Phospholipon 90H (100mg), cholesterol (10mg), and ethanol (35%) for cold, ethanol injection, and rotary evaporation methods. Developed ethosomes were assessed comparatively for vesicle size, zeta potential, PDI value, and % entrapment efficiency (EE). Based on the results of comparative evaluation; effective method was considered for optimization of parameters with respect to process and excipients concentration. 

 

Preliminary screening for development of TH ethosome:

Method for fabrication of ethosome was optimized from the following methods.

(a)    Cold method using magnetic stirrer (M1): PL90H, cholesterol, and TH altogether were dissolved in ethanol (organic phase) in a covered vessel at room temperature with vigorous stirring and further heated to 40°C in a water bath. The water was also heated to 40 °C in a separate vessel. The organic phase was added drop-wise to water phase with constant stirring on the hot plate magnetic stirrer at 700 rpm for 1 h in a covered vessel, keeping temperature constant at 40°C throughout the experiment^8. Ethosomes formed spontaneously were sonicated for 30 min using ultra-bath sonication.

 

(b)   Ethanol injection using homogenizer method (M2):

The organic phase constituting PL90H, cholesterol and TH was dissolved in ethanol in a covered vessel at room temperature with vigorous stirring. The resulting solution was maintained at 40°C in water bath. The water was heated to 40°C in a separate vessel.  Organic phase was added drop wise to water phase through injection with constant stirring at the center of vessel using homogenizer at 700 rpm for 1 h in a covered vessel, keeping temperature constant at 40°C throughout the study³. Further, developed multilamellar ethosome converted into unilamellar by sonication for 30 min on ultra-bath sonicator.

 

(c)    Mechanical dispersion using rotary flash evaporator (M3):

The TH-loaded ethosomes were prepared using a mechanical dispersion with the TFH method on a rotary flash evaporator. Briefly, PL90H, cholesterol, and TH at a certain amount were dissolved in a 5 mL of ethanol in a 25 mL round bottom flask. Ethanol was evaporated under reduced pressure over 15 min at 60 rpm to yield a uniform drug-lipid film inside the flask¹². The residual ethanol was volatilized thoroughly from the flask by keeping it in a desiccator overnight. The dried drug–lipid film was rehydrated with hydroethanol by rotating at 100 rpm for 1 h. The homogeneous TH-ethosomes suspension formed was sonicated for 30 min¹².

 

Optimization of formulation and processing variables of TH-loaded ethosomes:

Depending upon preliminary screening and results of comparative assessment, method M3 demonstrated relatively better results for vesicle size, zeta potential, and drug entrapment than methods M1 and M2. Therefore, method M3 was selected for studying impact of formulation and processing variables on development of ethosome. Concentration of ethanol, and PL90H showed a pronounced impact on formulation features and hence these varied simultaneously to study quantification of effect produced along with possible interactions¹².

 

Concentration of TH: PL 90H

The ratio of TH: PL90H significantly influences the physicochemical properties and skin permeation kinetics of ethosome. Literature reported suggests that phospholipids may mix with the stratum corneum lipid creating a lipid enriched environment¹³. This lipid enriched layer in the skin causes uptake of lipophilic drugs such as TH and enhances its bioavailability. In addition, phospholipid disturbs skin lipid layer and permits vesicles in deep skin layers. The concentration of phospholipid highly influences the formation of vesicles and drug loading¹³. Therefore, concentration of PL90H selected for the study was 0.5%, 1%, 1.5%, and 2% to focus a wide range of drug: lipid ratio viz., TH: PL90H as 1:2, 1:4, 1:6, and 1:8 and to assess its impact on vesicle size, PDI, zeta potential and EE%. The TH: PL90H ratio at values lower than 1:2 and higher than 1:8 was not considered, as was unable form uniform thin drug-lipid film during preliminary screening.

 

Effect of Ethanol:

According to the reported study, the ethanol used in hydration phase of ethosomes has a potential to enhance skin permeation by modification of physicochemical properties of vesicles and promote access across the stratum corneum¹³. The higher content of ethanol in ethosomes cause leakage of drug from vesicles and may result in severe skin irritation¹⁴. Therefore, concentrations of ethanol in the optimum ranges were evaluated for formulation of ethosomes¹³.

 

(a)    Volume of ethanol for film formation:

The volume of ethanol affects the formation of uniform film inside round bottom flask. The lower volume of ethanol than optimum may result in non-uniform film due to an insufficient amount of ethanol for spreading¹². Smaller amounts of ethanol may be evaporated rapidly that leads to uneven film distribution. Large volume of ethanol is not advantageous as it does not ensure complete removal of ethanol and leaves slight excess of residual ethanol and acquire more time for film formation and drying. Considering these preliminary parameters 5mL, 10mL, and 15mL volumes of ethanol were used to study film formation characteristics.

 

(b)    Concentration of hydroethanol for rehydration: The skin permeation is enhanced by the ethanol added during rehydration phase of ethosomes that alters the physicochemical properties of vesicles. Literature reports that more than 45% ethanol causes leakage of drug from vesicles and may result in severe skin irritation⁹. Therefore, concentration of ethanol for rehydration is preferred in the range 20-45%. Concentration <20% produces large vesicles and poor drug entrapment where as >45% leads to leakage of vesicle because of increased fluidity of vesicle bilayer^15,16. Therefore, ethanol concentration in hydroethanol for film rehydration was selected was 20%, 30%, 40%, and 45%.

 

Stirring speed on film formation:

Uniform the thin film formation is the prerequisite for identical development of vesicles. Film formation depends on the stirring speed of round bottom flask of rotary flash evaporator. Appropriate speed of stirring forms a uniform film; high speed evaporates solvent rapidly resulting in uneven film and low speed takes much longer time for development of film that leaves traces of solvent in film⁹. Considering these fact, desired stirring speed for film formation was evaluated at stirring rates 40, 60, 80, and 100 rpm.

 

Stirring speed for rehydration:

The vacuum dried film was rehydrated using hydroethanol solution as a rehydrating medium at a specified speed that influence formation of multilamellar vesicles⁹. Maximum stirring speed allowed at rotary evaporator was 100rpm and stirring speed was 60, 80, and 100 rpm. Stirring speed <60rpm was not considered as it forms large size vesicles. The effect of stirring speed for rehydration was studied on vesicle size, zeta potential, and PDI.

 

Sonication time: 

The vesicles prepared by TFH were multilamellar and reduced to nano-sized unilamellar by sonication for ease of penetration through skin⁹. In the present study, ultra-bath sonication was used. The optimum time for sonication was evaluated for processing time 15, 30 and 45 min; and its impact on vesicle size and EE%. Sonication time <15 min was not sufficient to break multilamellar vesicles in unilamellar and >45 min sonication breaks vesicles causing drug leakage.

 

CHARACTERIZATION OF ETHOSOME:

Vesicular size distribution, PDI and zeta potential:

The size distribution of ethosomes along with PDI, and zeta potential was measured by Dynamic Light Scattering (DLS) technique on instrument Malvern autosizer 5002¹⁷. The vesicular suspensions were mixed with the appropriate medium (PBS, pH 7.4) and the vesicle size, PDI and zeta potential were recorded in triplicate. Morphology of vesicles primarily observed under Motic instrument at 40x magnification.

 

Entrapment Efficiency:

The ultracentrifugation technique was utilized for the separation of the unentrapped drug from the developed ethosomal suspension. Accurately 2mL ethosomal suspension was diluted with Phosphate buffer pH 7.4 up to 5mL and centrifuged at 15,000 rpm for 1 h at 4°C using a cooling centrifuge. After centrifugation, the supernatant layer was filtered and analyzed on UV-VIS double beam spectrophotometer (Chemito Spectra scan UV 2600, India) at 223 nm^15,17. The percentage drug entrapment was calculated using eq. (1).

               

                 Drug Content

EE% = ------------------------ X 100………………….. (1)

                 Total Drug added

 

RESULT AND DISCUSSION:

Vesicular size distribution, PDI and zeta potential:

Vesicles prepared by M1, M2 and M3 demonstrated an insignificant difference in vesicle size 129.07±1.24nm, 115.4±1.82nm and 126.6±1.55nm, respectively. The ethanol has potential to reduce thickness of membrane and vesicle size probably due to formation of phase with interpenetrating hydrocarbon chain⁹. The microscopic evaluation indicated that all formulations contained spherical ethosomes under microscope Motic at 40x. Formulation processed by method M3 showed uniform spherical vesicles compared to M1 and M2 formulations. Besides, broken vesicles were observed more for M1 and M2 formulations that reduced the drug entrapment. M3 ethosome formulation exhibited uniform morphological features Fig. 1.

 

Fig. 1 Vesicle size (nm) of TH ethosome prepared by thin film hydration method (M3)

 

Polydispersity index (PDI):

PDI index (heterogeneity index) is used to measure distribution of molecular mass in polymer that indicates broadness of molecular weight distribution¹⁸. The larger the value the broader is the molecular weight. The standard PDI value is considered to be <0.5 and higher values indicates aggregation of particles¹⁰. All formulation exhibited PDI values in the range 0.2 to 0.4 indicating narrow distribution and homogeneity of size within the formulations. The lowest value of PDI 0.2±0.01 is observed for ethosome prepared using a M2 while 0.3±0.03, 0.4±0.04 for M1 and M3 formulations, respectively.

 

Zeta potential:

The charge on ethosome vesicle is an important constraint that influences stability as well as skin vesicle interactions⁹. During storage, the aggregation between vesicles can be prevented by electrostatic repulsion between charged vesicles. Ethanol concentration imparts a negative charge to the vesicles that prevent fusion of vesicles and affect skin permeation. Ethanol provides concentration-dependent negative charge to polar head of phospholipid that develops electrostatic repulsion and reduces vesicle aggregation¹⁹. Zeta potential of vesicles is significantly affected by drug, phospholipid and cholesterol; and interaction with ethanol¹⁹. In general zeta potential values more than -30 mV or +30 mV are considered to be stable^8,19. The zeta potential value of human skin is around +23 mV; so that negative zeta potential enhances adherence of vesicle to skin that is responsible for enhanced skin permeation. The highest zeta potential recorded was -56±2.3 mV for M3 formulation, Fig. 2. Ethosomes prepared by method M3 shown adequate electrostatic repulsion that prevents aggregation of vesicles and exhibits high stability. On other hand, method M1 formulation showed zeta potential -34.1±0.5 mV, which ensures stability of ethosome without aggregation. The method M1 formulation showed zeta potential -19±0.3 mV which is associated with poor formulation stability. Formulations processed using M3 showed highest zeta potential and deliberated as the most stable formulation.

 

Fig. 2 Zeta potential of TH ethosome developed by M3 method

 

Entrapment Efficiency:

The potential of vesicular drug delivery system is evaluated from the drug entrapment within the vesicular carrier. EE% is greatly influenced by concentrations of phospholipid and ethanol. The higher entrapment may be explained by multilamellarity of vesicles and high concentration of ethanol that causes thinning of bilayer membrane and makes vesicles more permeable leading to leakage of drug²⁰. The development of desired formulation is dependent not only on the excipients but also on drug and significantly on the method of preparation²⁰. In the present investigation, formulation M3 reported highest entrapment 82.10±0.25% as compared to methods (M2) 50.57±0.34%, and (M1) 59.55±0.21%. This implies that, method of preparation affects the entrapment of vesicles on high magnitude than vesicle size and zeta potential. In case of method M1 there was a chance of drug loss by evaporation that take place during injecting solution at a specific rate that decreases entrapment of drug within vesicles; that leads to formation of broken vesicles and decreased drug entrapment. Ethosome developed with method M2 indicate that during heating conditions at 40°C it may result in drug loss and less entrapment. Mechanical dispersion that constitutes formation of thin film of phospholipid with drug and during hydration it ensures maximum vesicle entrapment with minimum drug loss and uniform vesicle formation. The highest drug entrapment, appropriate vesicle size, and zeta potential are observed for formulation M3. Therefore, this method was selected as the most effective method for fabrication of TH ethosome.

 

The results of preliminary screening indicated that vesicle size, PDI values of M1, M2, and M3 formulation were significant for transdermal enhancement and physical stability of vesicles during storage. The zeta potential and EE% was recorded highest with M3 formulation as compared to M1, and M2 formulations. Therefore, it can be concluded that M3 ethosome with small vesicle size, unilamellar spherical shape with highest entrapment, and physical stability prepared by thin film hydration method (M3) was considered as an effective method for fabrication and optimization of TH loaded ethosome.

 

Optimization of Method M3

In the present investigation Method M3 was utilized to develop TH loaded ethosome; and excipients and process parameters were evaluated. The optimization included for process parameters were film formation stirring speed, film rehydration stirring time and sonication time; and for excipients it was concentration of TH: PL 90H, volume of ethanol for film formation and rehydration.

 

Concentration of TH: PL 90H

Concentration of TH: PL 90H was studied at concentrations 1:2, 1:4, 1:6, and 1:8 for a comprehensive understanding of the impact at low and high levels of ethanol concentration (20% to 40%)⁹. The effect of TH: PL 90H on vesicle size was mainly attributed to an increase in magnitude of the ratio that increases packing of phospholipid which broadens the thickness of a hydrophobic portion of bilayer and reduces the rate of motion of the lipid tails, leading to increased size of vesicles⁹. The results revealed that higher amounts of TH: PL 90H and ethanol reduced vesicle size proportionately signifying the dominating outcome of ethanol and increased zeta potential indicating stability of vesicles with homogeneity amongst vesicles demonstrated by evidence of low PDI, Table 1¹⁹. It was observed that at respective TH: PL 90H and ethanol concentration 20% to 40%, EE% was increased and could be due to the cosolvent effect of ethanol that facilitated loading of drug in aqueous core of the ethosome²⁰. The formulation B40 with vesicle size 127.39±3.61 nm, PDI 0.350±0.01, EE% 88.05±0.37, and zeta potential -45.5±3.25 was selected as optimized formulation based on smaller vesicle size; and good EE and zeta potential.

 

 

Table 1: Optimization of TH: PL90H for development of ethosomes

 

Table 2: Effect of sonication time on ethosome formulation

Formulation Code	Vesicle Size (nm)		Zeta Potential (mV)		PDI
	Sonication time (min)		Sonication time (min)		Sonication time (min)
	15 min	30 min	15 min	30 min	15 min	30 min
TH:PL 90H (1:2)	199.2±1.61	105.56±2.77	-30	-31	0.409±0.03	0.433±0.0029
TH:PL 90H (1:4)	320±1.06	136.21±2.81	-14	-23	0.313±0.012	0.376±0.009
TH:PL 90H (1:6)	430±2.03	178.71±2.07	-11	-24	0.652±0.032	0.434±0.011
TH:PL 90H (1:8)	510±1.16	348.32±1.2	-9	-16	0.898±0.056	0.52±0.011

 

Effect of ethanol:

(a)    Volume of ethanol for film formation:

The film formation characteristics were evaluated for 5 mL, 10 mL, and 15 mL volume of ethanol respectively. A 5 mL ethanol was not sufficient for uniform film formation for the specified drug: lipid and at higher lipid content it was unable to form a film. On the contrary, 10 mL ethanol attributed to uniform film formation for all drug: lipid ratios; while for 15 mL ethanol takes a longer time for film formation with uneven film with traces of residual ethanol. Hence, 10 mL ethanol was optimized for 1:2, 1:4, 1:6, and 1:8 drug: lipid ratio.

 

(b)    Ethanol in hydroethanol for film rehydration: The amount of ethanol in hydroethanol has a great influence on vesicle size, zeta potential, and EE%. The amount of ethanol in hydroethanol at about 20% to 40% at respective ratios of PL 90H: TH demonstrated a decrease in vesicle size with increase in entrapment efficiency²¹. At 45% ethanol hydroethanol resulted in reduced vesicle size but increases fluidity of lipid bilayer that leads to the leakage of vesicle and ultimately the % EE decreases. Hence, it can be stated that in the development of ethosomes concentration of ethanol in hydroethanol should be within 20% to 40% and was considered as optimum for development of TH ethosomes.

 

Effect of stirring speed on film formation:

The uniform film formation with complete removal of solvent is the prerequisite for the development of uniform vesicles formation⁹. The film formation characteristics were evaluated at speed 40, 60, 80rpm, respectively, Uniform film formation ensured at 60rpm with negligible traces of solvent. At stirring speed 40 rpm the rate of solvent evaporation was very slow that leaves traces of solvent with uneven film formation.  At high speed (80 rpm and 100                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                rpm) rate of solvent evaporation was too high that fails to form a film, hence 60rpm was selected.

 

Stirring speed for film rehydration:

The stirring speed for rehydration of dried film significantly affects vesicle formation. The lower speed 60rpm was insufficient to separate vesicles and network of vesicles formed, while maximum speed generated dispersed vesicles, for this reason, appropriate speed is important²¹. At 100rpm there was a higher shear rate at the interface of two phases. The vigorous, uniform and increased mechanical shear developed resulted in rapid division of formed giant vesicles and reduced coalescence and thus formed nano-sized vesicles²².

 

Effect of sonication time

The multilamellar vesicles formed after film rehydration was converted into the unilamellar vesicles using ultra-bath sonication. This technique induces pressure stress that breaks up large multilamellar vesicles to unilamellar with size ranging from 5-50nm²³. Hence, a short time of sonication was unable to break giant vesicles while longer duration of sonication affects the vesicle size because it breaks the membrane and form smaller vesicles.  

 

The time of sonication affects the vesicle size, zeta potential, and PDI presented in Table 2. The ultra-bath sonication for 15 min with 3 cycles of 5 min with rest cycles of 1 min were unable to break multilamellar vesicles and form large vesicle with aggregation, and turbid appearance of suspension that indicated insignificant zeta potential, and PDI values. Sonication for 30 min yields a clear dispersion indicating formation of unilamellar homogenous vesicles; and vesicle size and PDI²⁴. At 45 min sonication time, the smaller vesicles were formed but over a long period of sonication reduce ethanol content of ethosomes by evaporation and thus sonication time 30 min was selected as optimized one. (Table-2).

 

CONCLUSION:

The present aforementioned studies revealed that, terbinafine HCl ethosomes could be effectively prepared by mechanical dispersion thin film hydration method using rotary flash evaporator as compared to other methods studied. The effective development of ethosome essentially depends upon both; the formulation and processing parameters. The TH: PL90H ratio and ethanol 20 to 40% are dominating parameters that affect vesicle size, zeta potential, PDI, and % entrapment efficiency. The concentration of PL 90H and hydroethanol from 0.5 – 1.5% w/v and 20-40%, respectively, considered appropriate for fabrication of TH-loaded ethosomes. Stirring speed 60 rpm for film formation, 100 rpm film rehydration and 30 min bath sonication were appropriate at specific TH: PL90H ratio for effective vesicle size, zeta potential, and PDI and % entrapment efficiency.

 

ACKNOWLEDGEMENT:

The authors wish to thank Lipoid, Germany and FDC Pvt. Ltd. India, for supplying Phospholipon 90H and Terbinafine HCl as gift sample, respectively. The authors are thankful to Dr. H. N. More, Principal Bharati Vidyapeeth College of Pharmacy, Kolhapur, India and its other stakeholders providing timely help, guidance and research amenities.

 

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Received on 18.01.2021           Modified on 21.02.2021

Accepted on 12.03.2021          © RJPT All right reserved