international journal of pharma and bio sciences issn … j pharm bio sci 2013 oct; 4(4): (p) 497 -...

Int J Pharm Bio Sci 2013 Oct; 4(4): (P) 497 - 512

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Research Article Novel drug delivery system

International Journal of Pharma and Bio Sciences ISSN

0975-6299

RHEOLOGICAL INVESTIGATION OF NYSTATIN LOADED LIPOSOMAL

GEL FOR TOPICAL APPLICATION: FACTORIAL DESIGN APPROACH

SACHIN S. SALUNKHE*1, NEELA M. BHATIA 1

AND MANISH S. BHATIA 2

1

Department of Quality Assurance, Bharati Vidyapeeth College of Pharmacy,

Shivaji University, Near Chitranagari, Kolhapur, 416 013, Maharashtra, India. 2

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy,

Shivaji University, Near Chitranagari, Kolhapur, 416 013, Maharashtra, India.

ABSTRACT Liposomal carriers, eminent for their potential in topical drug delivery have been chosen to help embedding of nystatin molecules in the skin layers. The objective of present study was to develop and carry out rheological investigations of nystatin liposomal gel by using optimization technology in terms of 23 factorial design. In this study attempts have been made to formulate liposomal dispersions and gel formulations by ethanol injection method for topical drug delivery. Present model demonstrates the significance of factors on responses in the liposomal gel formulation. Gels containing liposomes (optimized batch) were prepared in Carbopol® 934 NF and were characterized for rheology, permeation and drug deposition. Factorial design of present study showed that dependent variables are the results of influence of independent variables.The results indicate that the utility of liposomal system as a vehicle for topical delivery of nystatin through the gel is excellent and rational. KEYWORDS: Factorial design, skin permeation, ethanol injection, rheology, liposomal gel.

SACHIN S. SALUNKHE Department of Quality Assurance, Bharati Vidyapeeth College of Pharmacy,

Shivaji University, Near Chitranagari, Kolhapur, 416 013, Maharashtra, India.



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INTRODUCTION

Polyene antibiotics are a class of biologically active bacterial metabolites isolated from Streptomyces, an aerobic actinomycetes genus obtained from soil. While more than one hundred polyene antibiotics have been described, amphotericin B (AmB) and nystatin are the two agents most commonly used to treat fungal and protozoal infections in humans1. AmB has been the most common antifungal agent used to treat invasive systemic fungal infections in critically ill patients over the past 50 years. This can be attributed to its wide spectrum of activity towards pathogenic fungi2,3. However, several side effects, especially nephrotoxicity and hepatotoxicity, have limited its use. In order to minimize these problems, various liposomal formulations were developed. In contrast to AmB, the use of nystatin is currently limited almost exclusively to the topical treatment of superficial Candida infections, since it is not effective when given orally and is severely toxic as an intravenous application4. Nevertheless, nystatin has recently been successfully incorporated into liposomal pharmaceutical formulations, which reduced its toxicity dramatically without losing its therapeutic properties5,6. It has been shown that the antifungal activity of nystatin is broader in comparison to AmB and could, therefore, be more effective when given by the intravenous route7. The polyene especially nystatin has high affinity for ergosterol present in fungi cell membrane, combine with it, get inserted into the cell membrane and several molecules together orient themselves in such a way as to form a micropore. The hydrophilic side forms the interior of the pore through which ions, amino acids and other water soluble substances move out. The micropore is stabilized by membrane sterols which fill up the spaces between these molecules on the lipophilic side – constituting outer surface of the pore and thus cell permeability is markedly increased1.

The hypothesis of this research is that the use of triethanolamine can neutralize the acidic carbopol dispersion, which will help in the stability of gel. In addition it may give a gel of suitable physical properties, high viscosity,

good permeability and consequently good bioavailability8. Carbopols belong to the class of synthetic high-molecular-weight polymers of acrylic acid that are crosslinked with either allyl sucrose or allyl ethers of pentaerythritol. They contain between 52 and 68% of carboxylic acid (COOH) groups calculated on the dry basis9. The carbopol molecule often exists as a strongly coiled spiral form. The unwinding of this spiral structure upon hydration leads to increase in viscosity. The unwinding of the carbopol resin may be explained by one of the mechanisms as described below. The most common mechanism is based on use of appropriate base for neutralization of the polymer. This neutralization imposes ionization of polymer leading to generation of negative charge on the polymer chains. Unfolding of the structure thus occurs through repulsion between these charges that on intertwining forms a three-dimensional matrix resulting in instantaneous formation of a highly viscous gel10. A number of polymers have been investigated to develop gel forming systems and the rheological properties and stability of different gel systems have been also investigated11. However a nystatin liposomal based gel delivery system for topical application had not been studied and is not available commercially. Considering above mentioned facts attempts have been made to formulate liposomal based gel for topical delivery of nystatin. The primary objective of this study was to develop nystatin loaded liposomes by factorial design approach. The optimized batch was further used for formulating liposomal topical gel and characterization of its physical properties and rheological behaviour.The microstructure of the gel influenced the permeability, duration of effect and absorbability and thus has pronounced effect on overall therapeutic performance 8. To understand the influence of gel microstructure on pharmaceutical performance of the prepared gel rheological investigations, in vitro skin permeation of drug and ex vivo drug deposition studies have been performed in the present work.



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MATERIALS AND METHODS Nystatin was kindly provided by Cipla Ltd. Patalganga, Mumbai (India). Saturated phosphatidylcholine (PL; Phospholipon 90H) was a generous gift from Nattermann phospholipids GmbH,Germany. Cholesterol (CHL), Stearic acid (SA) was purchased from Qualigens Fine Chemicals, Mumbai, India and Research Lab, Mumbai, India, respectively. Reference marketed product (gel) was procured from local market. Carbopol® 934 NF (poly acrylic acid polymer) was gifted by Noveon, India. All other chemicals used were of HPLC or analytical grade. (i) Liposome preparation Aqueous liposomal formulations were prepared by ethanol injection method12.The phospholipid (phospholipon 90H), cholesterol, steric acid and nystatin were weighed proportionally and then mixed with 6mL ethanol. This solution was kept for sonication to get transparent solution. The resultant ethanolic solution was injected in preheated (60ºC) buffer solution at constant stirring with the help of syringe 8. The formed dispersion

was kept for further stirring for 30 min, furthermore prepared liposomal dispersion was allowed to mature at room temperature and then stored into 2-8ºC. After this heating was stopped and stirring kept for 20 min more to get the stable liposomal dispersion. The dispersion was allow to remain at room temperature for 2-3 hthrough the point of restructuring of liposomes by swelling behaviour. (ii) Effect of variables To study the effect of variables on liposome performance and characteristics, different batches were prepared using 23 factorial design approach. The 3 independent variables investigated were the amount of PL 90H (X1), CHL (X2), and SA (X3), respectively. The effect of the above mentioned independent variables was investigated on the dependent variables such as vesicle size (Y1) and entrapment efficiency (Y2). Amount of nystatin drug (20 mg) was kept constant. Values of all variables and batch codes are shown in Table 1. Single replicate at the centre of the design were investigated to allow for an independent estimation of the experimental design.

Table 1

Experimental design with coded levels of variables and actual values.

Batch code Variable X1 Amt. of PL 90 H (mg)

Variable X2 Amt. of CHL (mg)

Variable X2 Amt. of SA (mg)

LP1 60 (-1) 40 (+1) 40 (+1)

LP2 60 (-1) 40 (+1) 30 (-1)

LP3 60 (-1) 20 (-1) 40 (+1)

LP4 60 (-1) 20 (-1) 30 (-1)

LP5 80 (+1) 40 (+1) 40 (+1)

LP6 80 (+1) 40 (+1) 30 (-1)

LP7 80 (+1) 20 (-1) 40 (+1)

LP8 80 (+1) 20 (-1) 30 (-1)

(iii) Size distribution Prepared liposomal batches were monitored for their morphological attributes using optical microscope13. Mean vesicle size and size distribution profile of liposome was determined by dynamic light scattering using Malvern Hydro 2000 SM particle size analyzer (Malvern Instruments, Malvern, UK) at room temperature which follows Mie's theory of light scattering. Before measurement, liposomal batches were diluted with filtered bi-distilled water. The average hydrodynamic particle

size and polydispersity index were (PI) of diluted dispersions were measured.

Zeta potential (ζζζζ) determination Charge on empty and drug loaded vesicles surface was determined using Zetasizer 300HSA (Malvern Instruments, Malvern, UK). Analysis time was kept for 60 s and average zeta potential and charge on the liposome was

determined. The ζ potential was measured after dilution of samples with distilled water at room temperature.



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(iv) Entrapment efficiency Entrapment efficiency of nystatin loaded liposomes were measured by gel permeation chromatography. Entrapment efficiency was determined by mini-column centrifugation method14.Sephadex® G25M solution (10%, w/v) was prepared in double distilled water (DDW) and was kept aside for 24 h for swelling. To prepare mini-columns, Whatman filter pad was inserted in 1mL syringe and swelled Sephadex was added slowly to it. Care was taken to avoid air entrapment in the column. Excess amount of water was removed by spinning the column at 3000 rpm for 5 min using Eppendorff centrifuge 5810 R (Hamburg, Germany). Liposomal formulation (100 µL) was slowly added on prepared column and centrifuged as earlier. Procedure was repeated on the same column by adding 100 µL of DDW. Free drug remained bound to the gel, while entrapped micro-particulate drug travelled through the gel and was collected from first and second stage of centrifugation. Obtained eluted liposomal samples were ruptured using sufficient volume of methanol and the absorbance of the ruptured eluents was measured at 305 nm with a UV spectrophotometer. Each determination was run in triplicate15. Percent entrapment was calculated from total amount of nystatin present in 100 µL of liposomes by UV (ultraviolet) spectrophotometer. Using Equation 1, method was validated by applying free drug solution instead of liposomes16. Entrapment efficiency (EE) = (Qe/Qt) × 100 (1) Where Qe is the amount of entrappednystatinand Qt is the amount of nystatin present in 100 µL of liposomes. (v) Skin permeation and drug deposition

studies The male wistar rats were kept in animal house under standard laboratory conditions with free access to diet and water as per guidelines of CPCSEA. Male wistar rat was sacrificed by exposing to excess of chloroform. To the abdominal skin, depilatory (Anne French, India) was applied and kept for 15 min to remove the hair from the skin. After 15 min of application, skin was washed with water. Skin was excised from rat with scalpel

and fatty layer was removed by keeping the skin in warm water at 37±2ºC. After 5 min, fatty layer was peeled off gently and skin was washed with water and kept for saturation in phosphate buffer saline pH 7.4 for about 30 min before it was used for permeation studies. Skin permeation studies with nystatin containing liposome formulations were carried out using abdominal rat skin, employing modified Franz-diffusion cells17. The results obtained were compared with that of non-liposomal formulations of nystatin. The skin was prepared by mounting on the receptor chamber with cross-sectional area of 3.14 cm2 exposed to the receptor compartment. The receptor compartment was filled with (22 mL) phosphate buffer pH 7.4. It was jacketed to maintain the temperature 37 ± 0.5 º C and was kept stirring at 100 rpm. Prior to application of formulations, the skin was allowed to equilibrate at this condition for 1h. Liposomal or non-liposomal nystatin formulation (amount equivalent to 5 mg of drug) was applied uniformly on the dorsal side of skin. Aliquots of 2 mL were withdrawn periodically and replaced with same amount of saline solution to maintain the receptor phase volume at a constant level. The samples were quantified spectrophotometrically at a λmax of 305 nm. For determination of drug deposited in the skin, cell was dismantled after a period of 24 h and skin was carefully removed from the cell. The formulation applied on skin surface was swabbed first with phosphate buffer pH 7.4 and then with methanol. The procedure was repeated twice to ensure no traces of formulation are left onto skin surface. Drug present on the surface of the skin was removed by using Scotch Tape (Scotch Magic Tape, 810, Birla 3M Ltd., Bangalore, India)18. After stripping the skin was cut into small pieces and drug present in the skin was extracted in methanol under sonication and estimated by UV spectrophotometer after suitable dilution and filtration. (vi) Preparation of liposomal gel On the basis of factorial design approach, liposome batch (LP6) was selected for further formulation studies of liposomal gel. Gel was prepared using carbopol® 934 NF (1, 1.5 and 2%w/v). The appropriate quantity of carbopol



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934 powder was dispersed into distilled water under constant stirring with a glass rod, taking care to avoid the formation of indispersible lumps and allowed to hydrate for 24 h at room temperature for swelling. Topical liposomal gel formulations were prepared by incorporation of liposome's containing nystatin (separated from the unentrapped drug) were mixed into the carbopol gel with a mechanical stirrer (50 rpm, 5 min). The addition of triethanolamine (0.5% w/w) was used for neutralization of whole dispersion which imparts for integrity of gel by equalizing acid base balance. Control gels were made under the same conditions 19,

20. (vii) Rheological studies While considering the development of stable liposome dispersion for topical delivery system, they usually need to be incorporated into convenient suitable dosage form to obtain formulation with desired semisolid consistency and rheological behaviour. It controls the flow properties to ensure product quality and effectiveness during formulation of respective preparation. It helps in selection of dermatological formulation that will progress to clinical efficacy. In present study liposomal gels were prepared using carbopol 934 as gelling agent. Rheological analysis of liposome loaded carbopol gels of nystatin were performed using a stress control rheometer (ViscotechRheometer, Rheologica Instruments AB, Lund, Sweden), equipped with stress rheologic basic software, version 5, using cone-plate geometry with a diameter of the cone being 25 mm and a cone angle of 1º, operating in the oscillation and static mode. Rheological analysis was performed at room temperature. The following parameters were carried out for rheology measurement. Oscillation stress sweep Dynamic oscillation stress sweep was performed to determine the linear viscoelastic region (LVR). The region of stress at which elastic modulus (G’) is independent of applied stress known as LVR. Over the range of LVR, no significant microstructural change occurs in polymer. This test gives idea about the critical stress beyond which the sample may show significant structural changes, and therefore

the consequent choice of the stress value to be used in other oscillation tests. The samples were exposed to increasing stress (0.1 to 100 Pa) at a constant frequency of 0.1 Hz 21. Oscillation frequency sweep The samples were exposed to stepwise increasing frequency (0.1 to 100 Hz) at a constant stress in the field of LVR and elastic modulus (G’) as well as viscous modulus (G″) were recorded against frequency22. Creep-recovery The creep recovery test was used to determine the viscoelastic properties of the optimized samples of gel23. The samples were exposed to the selected averaged stress of the stress sweep mode for 50 s. It was followed by relaxation period for 100 s for recovery. The creep compliance Jc (defined as the ratio of measured strain to the applied stress) was monitored against time. (viii) Drug content and content uniformity The gel sample (100 mg) was withdrawn and drug (nystatin) content was determined using UV spectrophotometer at 305 nm. For determination of drug content methanol was used to extract the drug and same procedure was utilized for content uniformity of drug from whole batch of formulation (ix) Stability studies The ability of vesicles to retain the drug (i.e. drug retentive behaviour) was assessed by keeping the liposomal suspensions and liposomal gel at two different temperature conditions, i.e. 4-8 °C (Refrigerator; RF),25±2 °C (Room temperature; RT), for a period of 60 days. Samples were withdrawn periodically and analyzed for the particle size and entrapment efficiency as previously described 24.

RESULTS AND DISCUSSION

Amount of PL 90H, CH and SA were found to be crucial in the preparation and stabilization of liposomes and hence selected as independent variables in the 23 factorial designs (Table 1). In a preformulation study the optimum concentrations of PL 90H, CH,



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and SA were determined to obtain stable liposomes free from aggregation, fusion and sedimentation. Liposomes were prepared using ethanol injection method and hence this technique was found to be well suited for the

production of liposomes without aggregation. Responses as dependent variables of different batches were obtained by using factorial design (Table 2).

Table 2

Results obtained for all experimental batches by using factorial design.

Batch code Vesicle size (µm) Mean ± SD

Polydispersity Index (PdI) Mean ± SD

Zeta Potential (-mV) Mean ± SD

Entrapment Efficiency (EE %) Mean ± SD

LP1 3.96 ± 1.38 0.632 -52.3 ± 1.45 66.15 ± 1.74

LP2 4.25 ± 0.95 0.598 -52.1 ± 0.63 65.35 ± 0.74

LP3 4.44 ± 0.30 0.430 -57.9 ± 1.51 64.88 ± 0.83

LP4 6.02 ± 1.39 0.731 -51.3 ± 1.49 55.18 ± 0.50

LP5 5.60 ± 1.40 0.331 -58.6 ± 1.19 62.56 ± 0.45

LP6 3.01 ± 1.25 0.231 -63.6 ± 1.41 73.40 ± 0.17

LP7 5.79 ± 1.58 0.410 -58.9 ± 1.87 63.90 ± 1.59

LP8 3.50 ± 0.91 0.348 -54.2 ± 1.48 66.20 ±1.50 * Mean ± SD, n=3.

Obtained data was subjected to multiple regression analysis using STATEASE (Design Expert Trial, Version 8.0) software and obtained data were fitted in Equation 2. Y=B0 +B1X1 + B2X2 + B3X3 + B12X1X2 + B13X1X3 + B23X2X3 + B123X1X2X3 (2) (i) Effect of variables on particle size The most significant parameter, which needs to be monitored during liposome preparation is the vesicle size and size distribution of liposomes. From literature, it was observed that the size distribution of the liposome determines their in vivo or ex vivo performance 8. Some reports have shown that liposome size has remarkable effect on the drug release as well as drug deposition in the skin25. Thus for the effective delivery, the selected method should result in optimum size range. In the present study, ethanol injection method was found to produce polydispersity index in the range of 0.231-0.731which indicates narrow size distribution (Table 2). It was observed that the relative amount of PL 90H, CH and SA was found to play important role in formation of vesicle size (Figure 1).

Size of vesicles were found to be in the range of 3.01± 1.25 µm to6.02 ± 1.39µm. To understand the effect of lipid concentration on vesicle size, coefficients observed for liposomal size were fitted in Equation 3. Y1=4.57 – 0.096X1– 0.37X2+ 0.38X3+ 0.20X1X2+ 0.84X1X3+0.20X2X3–0.078X1X2X3

(3) An independent negative correlation

was observed for both the variables X1(PL 90H) and X2 (CH) while positive correlation for X3 (SA) in case of liposome vesicle size (Equation 3; r2 = 0.835) whereas interaction between the independent variables has shown positive correlation with respect to dependent variables. Thus with combined increase in the concentration of PL 90H, CH and SA; vesicle size was found to be increased. The observed coefficient values illustrated that independent variables have more prominent effect on dependent responses. The mean vesicle diameter of liposomal batch LP6 was found to be 3.01 ± 1.25 µm (Fig 1). The prepared liposomal formulations were further subjected to determine percentage EE.



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a.

b.

Figure 1 Effect of independent variables on response of vesicle size (µm):

a) Effect of amount PL 90 H and CHL b) Effect of amount of CHL and SA.

(ii) Effect of variables on entrapment efficiency (% EE) Percentage EE is expressed as the fraction of drug incorporated into liposomes relative to total amount of drug used. In the present study, the observed % EE for all batches were in the range of 55.18±0.50% to73.40 ± 0.17%. To understand the effect of lipid concentration on entrapment efficiency, coefficients were observed as in Equation 4. Y2=64.70+1.81X1+ 2.16X2– 0.33X3– 0.70X1X2–2.96X1X3– 2.18X2X3 – 0.63X1X2X3 (4)



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A positive correlation was observed for both variables X1 (PL 90H) and X2(CH) while negative correlation for X3 (SA). Thus with increase in the concentration of PL 90H and CH entrapment efficiency was found to increase (Fig 2).

a.

b.

Figure 2 Effect of independent variables on response of entrapment efficiency (% EE):

a) Effect of amount PL 90 H and CHL b) Effect of amount of CHL and SA.

Among all the batches, LP6 had minimum vesicle size and maximum EE of 73.40 ± 0.17%. Small vesicle size occupies uniform distribution of particles leads for maximum entrapment. Hence LP6 batch was selected for the further study of gel formulation. Small particle size liposomes can uniformly cover

greater area of skin surface in comparison to larger particle size liposomes. Also, the polydispersity index of LP6 was smaller as compared to all other liposome batches, which indicates narrow particle size distribution, All prepared Nystatin loaded liposomes further



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studied to know zeta potential present on the surface of liposomes.

(iii) Determination of zeta potential (ζζζζ)

In the present study, zeta potential(ζ) obtained for liposomes are shown in Table 2. The values of zetapotential (-51.3 ± 1.49 mV to -63.6 ± 1.41 mV) for vesicles indicate that the prepared liposomes have sufficient charge to avoid aggregation of vesicles. (iv) Rheological study Study of the rheological properties is very important because the microstructural environment or mobility and other gel properties can be indirectly probed using these measurements. The results of rheological tests can give information about their behaviour during production, storage and application. The rheological studies assist to explore the viscoelastic properties of gel system under study. Rheological studies can be performed by static (Rotational) and dynamic (Oscillatory) measurement mode. The dynamic rheology provides a more direct correlation with microstructure than steady rheology since the materials can be examined in their at rest state without causing any disruption of their underlying structures. The semisolid preparations should flow or deform after applying the force and regain its elasticity as the force is removed. Thus, to understand the rheological properties of liposomal gels and for selection of optimum concentration of carbopol for desired rheological properties, different concentrations (1, 1.5, 2% w/v) of carbopol 934 were used to prepare liposomal gels at 25 °C with neutralization method26. The rheologies of all samples were determined to identify the minimum concentration of carbopol required for the

formation of gel with good viscoelastic properties. Oscillation stress sweep In an oscillation stress sweep test the response of the material (strain) is measured while exposing the material to an increasing stress and a constant frequency. In the linear viscoelastic area the ratio of stress and strain is a function of time alone. The solid moduli (G’) represents the total energy stored in the elastic bonds of system while viscous moduli (G’’) represents total energy expelled from system. Stress sweep was carried out to determine the linear viscoelastic region, where G’ is independent of applied stress. LVR has great significance for frequency sweep structure as we have to be sure that the frequency sweep is carried out in ground state of the material i.e. there must not be any breakdown of the structure. During stress sweep, it was observed that there was linearity between stress and strain produced all over the applied stress range, which indicate that system is working in correct range. Liposomal Gel with 2% w/v of carbopol had shown maximum G’ and LVR as compared to other systems and it was comparatively more stable over applied stress range. In oscillatory stress sweep test, system with higher LVR and G’ are considered as more elastic27. Lower degree of dependence of solid modulus G’ was observed for 2% w/v gel and it suggests that gel is of higher strength. Both 1% and 1.5% w/v systems have presented lower G’ and LVR and higher degree of dependence of G’ over applied stress indicating formation of weak gel. For 2% system, G’ was greater than G’’ over all applied stress, while for 1% and 1.5% system, G’ was higher than G’’ at lower stress (Fig 3). Hence the trend of G’ >G’’ for 2% w/v gel is a supplementary proof for experimental results21.



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Figure 3 Stress sweep for G' and G'' of nystatin liposomal gel containing 2% w/v carbopol.

The phase angle is good indicator of viscoelastic nature of system, being measure of the lag in sine response after an oscillatory stress has been applied to the sample. For perfectly elastic system, phase degree must be close to 0° and for viscous system it must be close to 90°28. Both nystatin liposomal gels containing 1% and 1.5% w/v carbopol show phase transition at higher stress. These systems have presented the change of behavior from elastic to viscous one, thus proving the instability of the system at higher

stress. Therefore, from stress sweep, it was concluded that nystatin liposomal gel prepared with 2% w/v carbopol are more stable and elastic. Oscillation frequency sweep Figure 4, presents the behaviour of G' over the applied frequency range. It was concluded that 2% system had shown independent behaviour of G' over applied frequency, while 1 and 1.5% system have exhibited a monotonous increase in solid component.

Figure 4 Frequency sweep for phase degree.



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It is possible that monotonic increase in G' at higher frequencies means the partial breakage of the interconnected network, which represents a true cross-linked polymer gel network. It was concluded that the gel network was retained at low frequencies and on the other hand, destroyed by the more frequent changes of the displacement at higher frequencies due to the formation of too rigid and brittle structures22. It was clear that 2% system had shown low dependency on frequency for viscous moduli and phase

degree than system containing 1 and 1.5% system. The value of phase degree for 2% gel was well in the viscoelastic region while for 1 and 1.5% systems the values indicated weak breaking structure. In frequency sweep test a new terminology was introduced i.e. loss tangent (tan δ). It is the ratio of loss modulus (G'') and storage modulus (G'). The higher the loss tangent less elastic is the material. For liposomal gel containing 2% w/v carbopol, tan δ was clearly below 1, while for 1 and 1.5% system it was much higher than 1 (Fig 5).

Figure 5 Frequency sweep for tan θ.

With this perspective, only the 2% system is elastic and stable. In frequency sweep, there must be non-dependency of both G' and G'' over the applied frequency and condition of G'>G'' must be prevalent over complete range29. Considering all these criteria, liposomal gel containing 2% w/v carbopol emerged as stable and elastic gel system for the formulation. Creep recovery The creep-recovery percent for liposomal gel containing 1%, 1.5% and 2% w/v carbopol

was found to be 48.59%, 69.97%, 74.40%, respectively. Thus the 2% gel has highest elastic recovery, which is said to be highly stable and easy to process, store and apply system. The 2% system had lowest compliance value i.e. J suggesting higher elasticity (Fig 6)23. Therefore from creep recovery test, it was observed that all systems have shown considerable elastic and viscoelastic recovery but liposomal gel with 2% w/v carbopol had presented highest recovery and lowest compliance (J).



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Figure 6 Creep recovery test.

From oscillatory rheological measurement, it was observed that liposomal gel prepared from 2% w/v carbopol was highly elastic, stable and having high recovery capacity. From these rheological properties, it can be concluded that this system is having good flow properties, good spreadability and applicability. Hence, further studies were performed with liposomal gel with 2% w/v of carbopol. (v) Drug content uniformity and pH

measurement The method used for preparation of liposomal based gel gave satisfactory results for drug

content as there was no significant difference observed in the % drug from various locations. The pH of the developed formulations was in accordance with that of human skin pH rendering them more acceptable. Thus prepared liposomal gel was suitable for topical application. (vi) Skin permeation and drug deposition

studies The results of in vitro drug permeation and deposition profile from different formulations of nystatin were shown in Table 3.

Table 3

In vitro skin permeation and skin retention of nystatin from different formulations.

Formulation Mean cumulative drug permeated (µg/cm

2)

Permeation flux Jss (µg/cm

2/h)

% Drug deposited in skin

Liposomal dispersion 1280 ± 75* 8.43 ± 1.02* 17.73 ± 2.30**

Liposomal gel 1105 ± 76* 7.71 ± 0.89* 20.71 ± 1.50***

Marketed gel 953 ± 83 6.03 ± 0.91 10.2 ± 1.10 * Mean ± SD, n=3, Dunnett multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.

Significant escalation in the skin permeation of nystatin has been observed (Fig 7) with liposomal based gel formulation in comparison to liposomal dispersion and marketed gel formulation.



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Figure 7 Permeation profile of different nystatin containing systems.

It reveals that the amount of nystatin permeated through the excised rat skin after 24 hrs was found to be 1280 ± 75, 1105 ± 76 and 953 ± 83 µg/cm2 from liposomal dispersion, liposomal gel and marketed gel, respectively. Higher values of flux obtained with liposomal dispersion 8.43 ± 1.02 µg/cm2/h and liposomal gel 7.71 ± 0.89 µg/cm2/h, than that obtained with marketed gel 6.03 ± 0.91 µg/cm2/h, clearly assure permeation enhancing effect of vesiculation. Results of this study clearly depict that the amount of drug retained in the skin was considerably higher in case of liposomal based dispersion and gel preparations, than with reference product. This gave an understanding that liposomes could not only enhance the penetration of drug molecules but also helped to localize the drug in the skin17. It was observed that % of nystatin deposited in skin for liposomal gel was maximum (20.71 ± 1.50 %) while in case of liposomal dispersion and marketed gel formulation percentages observed were17.73 ± 2.30 and 10.2 ± 1.10 %, respectively. Improved skin permeation of nystatin along with its enhanced deposition in the skin with liposomal formulation can be attributed to the lipo-solublized state of nystatin molecules by embedding in liposome shell. This was achieved in the presence of

aqueous and non-aqueous phase of bilayered systems, a state most ideally suited for drug penetration. The liposomal phospholipids (also one of the natural constituent of skin lipids) helped in generating and retaining the required physico-chemical state of the skin for enhanced permeation of nystatin. The phospholipid rich domains of vesicles might have helped to produce the depot effect for drug moleculewhich has been reflected as higher amount of drug retention within the skin layers in case of liposomal formulations such as liposomal gel and dispersion. Thus, the liposomal based nystatin formulations, with desired characteristics for topical administration, could be successfully prepared as a novel delivery system. The formulated nystatin liposomes have shown significantly enhanced skin permeation as well as retention of drug molecules in the skin. (vii) Stability study Stability of liposome dispersion (LP6) as well as 2% w/v carbopol liposomal gel containing 0.5% nystatin was carried out for 60 days at 4-8°C and room temperature. Responses obtained for different parameters for liposomal dispersion (which was subsequently formulated into liposomal gel) during stability period are shown in Table 4.



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Table 4 Effect on vesicle size and entrapment efficiency for optimized batch

(LP6) of liposomal dispersion during stability.

Days Vesicle size (µm) Entrapment efficiency %

Room Temp. 4-8º C Room Temp. 4-8º C

0 3.01 ± 0.54 3.01 ± 0.67 73.40 ± 4.50 73.40 ± 5.34

30 3.80 ± 0.72 3.50 ± 0.53 71.56 ± 3.89 72.67 ± 3.23

60 4.10 ± 0.46 3.90 ± 0.76 69.56 ± 4.10 70.32 ± 4.35 * Mean ± SD, n=3.

LP6 liposomes were found to be stable in terms of aggregation, fusion and vesicle disruption tendencies, over the studied storage period. From results it can be concluded that at room temperature and freeze temperature there was slight but insignificantly decrease in % entrapment efficiency and increase in particle size for liposomal batch LP6. Results suggest that stability problems of liposomes would be overcome by keeping the liposomal product in refrigerated condition24.

CONCLUSION

Preparation of liposomal formulations using factorial design proved to be a sound approach to obtain stable optimized formulation of nystatin. Variables such as amount of (Phospholipon) PL 90H (X1), (Cholesterol) CHL (X2), and (Stearic acid) SA (X3) have a profound effect on the vesicle size and entrapment efficiency. Rheological studies of all nystastin liposomal gels prepared with 1%, 1.5%, and 2% w/v carbopol 934 NF gave a clear idea about required optimum concentration for ideal rheological behaviour. Liposomal dispersion and gels

were found to be significantly increase (over 25% increase for liposomal dispersion and 13% increase for liposomal gel) the skin permeation and deposition in comparison to standard reference product. Stability studies performed for liposomal dispersion (LP6 batch) indicates the prepared liposomes have more stability at freezing temperature than that at room temperature. Hence from results obtained, it can be concluded that liposomal gel containing nystatin has potential application in topical delivery.

ACKNOWLEDGEMENT

We are highly thankful to Principal, Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing necessary facilities to carry out research work. The authors are highly thankful to Cipla Ltd. Patalganga; Mumbai (India), Nattermann phospholipids GmbH; Germany for providing gift samples of nystatin and phospholipids. DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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