Solid Lipid Nanoparticles Bearing Flurbiprofen for Transdermal Delivery

Abstract

Topical application of the drugs at the pathological sites offer potential advantages of delivering the drug directly to the site of action and thus producing high tissue concentrations of the drug. The solid lipid nanoparticles (SLN) bearing flurbiprofen were prepared by microemulsion method by dispersing o/w microemulsion in a cold aqueous surfactant medium under mechanical stirring. The SLN gel was prepared by adding SLN dispersion to polyacrylamide gel prepared by using polyacrylamide (0.5%), glycerol (10%), and water (69.5%). Shape and surface morphology was determined by scanning electron microscopy that revealed fairly spherical shape of the formulation. Percent drug entrapment was higher in SLN dispersion in comparison to SLN gel formulations. In vitro drug release, determined using cellophane membrane, showed that SLN dispersion exhibited higher drug release compared with SLN gel formulations. Both the SLN dispersion and SLN-gel formulation possessed a sustained drug release over a 24-hr period, but this sustained effect was more pronounced with SLN-gel formulations. The percent inhibition of edema after 8 hr was 55.51 ± 0.26% in case of SLN-T4-gel, whereas flurbiprofen and SLN-T4 dispersion exhibited 28.81 ± 0.46 and 31.89 ± 0.82 inhibition of edema. The SLN-T4-gel not only decreased the inflammation to larger magnitude, but also sustained its effect.

Keywords :

• Flurbiprofen
• Solid Lipid Nanoparticles
• Transdermal delivery

Nanoparticles prepared from solid lipids at room temperature have been proposed as a new type of drug carrier system. Solid lipid nanoparticles (SLNs) could be established as an alternative particulate carrier system by various research groups (Almeida, Runge, and Muller 1997; Siekmann and Westesen 1992). Recently increasing attention has focused on these SLNs because as colloidal drug carriers they combine advantages of polymeric nanoparticles, fat emulsions, and liposomes but simultaneously avoiding some of their disadvantages (Boltri et al. 1995; Sjostrom and Bergenstahl 1992). Under optimized conditions, SLNs can be produced to incorporate lipophilic or hydrophilic drugs and seem to fulfill the requirements for an optimum particulate carrier system (Muller et al. 1995; Schwarz et al. 1994).

The advantage of SLN is that the lipid matrix is made from physiological lipids and polymers from natural and synthetic sources, which decreases the danger of acute and chronic toxicity. The choice of the emulsifier depends on the administration route and is more limited for parenteral administrations. Early work on SLN dispersions mainly focused on the parenteral route of the drug administration; sustained release and drug targeting were the primary objectives (Schwarz and Mehnert 1997; Heiati, Tawashi, and Phillips 1998). Recently peroral or dermal routes of SLN administration have been evaluated (Muller and Dingler 1998).

SLNs are nonirritant and nontoxic (Muller et al. 1996), hence they are well suited for use on damaged or inflamed skin. Sustained and controlled drug release properties of SLN also can be helpful for dermal formulations, e.g., for the topical application of antibiotics, irritant drugs like benzoyl peroxide (Wester et al. 1991), or tretinoin (Masini et al. 1993; Schafer-Korting, Korting, and Ponce-Poschl 1994).

SLNs also have been found to modulate drug release into the skin and to improve drug delivery to particular skin layers in vitro (Maia, Mehnert, and Schafer-Korting 2000). The loss of water after application on the skin causes changes in the lipid and SLN structure. Electron microscopy indicates that dense films are formed after drying (32°C) of SLN dispersions in contrast to spherical structure (Dingler et al. 1999). The formation of the dense structure favors occlusive effects on the skin. It is interesting to note that the films made from melts of the lipid bulk do not form close films as dried SLN dispersions performed.

In the present investigation, we aimed to develop SLNs bearing flurbiprofen for transdermal delivery, which effectively would manage the pain and inflammation in osteoarthritis and rheumatoid arthritis.

MATERIALS AND METHOD

FDC Ltd. Mumbai, India, generously supplied flurbiprofen as gift sample. Cholesterol, soya lecithin, pluronic F-68, and 6-carboxyfluorescein were procured from Sigma (St. Louis, MO, USA). All reagents were of analytical grade and water was double distilled.

Preparation of SLNs

SLNs loaded with flurbiprofen were prepared using microemulsion technique by dispersing warm o/w microemulsion in a cold aqueous medium under mechanical stirring (Cavalli et al. 1995). Microemulsion was prepared by adding flurbiprofen (100 mg) to melted lipid at 70°C followed by successive addition of surfactant (soya lecithin), co-surfactant (butanol), and warm water. A clear microemulsion was obtained easily under gentle stirring at ∼70°C (a temperature close to melting point of the lipid used). SLNs were obtained by dispersing the warm o/w microemulsion (∼70°C) in a cold aqueous medium of pluronic F-68 under mechanical stirring at a ratio of 1:10 (v/v) (microemulsion: aqueous medium). Then the formulation was passed through Sephadex G-50 column to separate unentrapped drug and sonicated. The resultant preparation was stored at 4 ± 1°C. The SLN dispersion (20%) was finally added into the polyacrylamide gel (prepared by using 0.5% polyacrylamide, 10% glycerol, and 69.5% water) and were transferred in a beaker and stirred.

Optimization of Formulation Components

Stearic acid/cholesterol ratio in the SLN dispersions was optimized by preparing it with 9:1, 8:2, 6:4, 5:5, 4:6, 2:8, and 1:9 w/w ratio of stearic acid and cholesterol, and other parameters were kept constant. The average particle size was determined using laser diffraction particle size analyzer (Cilas, 1064L, France).

Further, the lipid/surfactant ratio in SLN dispersions was optimized. The SLNs were prepared using soya lecithin:lipid ratio of 0.2:1 to 1:1 w/w in presence of water containing 6-carboxyfluorescein (CF), and the other parameters were kept constant. The soya lecithin-stabilized formulations were subjected to CF quenching study to maximize the SLN/liposomes ratio. The preparations were then passed through Sephadex G-50 column and centrifuged at 10,000 rpm for 35 min. Eluents from each tube were removed and discarded. PBS (5 ml) was filled in each column and centrifuged, as previously, to recover all eluents containing unentrapped CF. After suitable dilution, the concentration of the free CF was measured with photoflorimeter at an emission wavelength of 518 nm.

The pluronic F-68: lipid ratio was optimized in regard to average particle size and percent drug entrapment while other parameters were kept constant. The SLN dispersions were prepared with 0.2:1 to 1:1 w/w ratio of pluronic F-68 to lipid and average particle size and percent drug entrapment was determined.

The sonication time was optimized for getting small particles with maximum drug entrapment using optimum stearic acid/cholesterol ratio and pluronic F-68/lipid ratio with varying sonication time (2, 3, 4, 5, 6, 7, and 8 min). The SLN dispersion (SLN-T4), prepared using optimized stearic acid/cholesterol, pluronic F-68/lipid ratio, and sonication time, was incorporated into a gel for transdermal application. These gels were characterized for percent drug entrapment, in vitro drug release, and in vitro drug skin permeation studies. Another three formulations (SLN-T1, SLN-T2, SLN-T3) also were taken for comparative study.

In Vitro-Characterization

The SLNs were characterized for various physicochemical attributes. The shape and surface morphology of SLN were visualized under scanning electron microscope (Leo VP 435 electron microscope). Particle size and polydispersity index of SLN dispersions were measured by laser diffraction-based particle size analyzer (Cilas) after suitable dilution. The surface charge of SLN was determined by the electrophoretic mobility of SLN in a U-type tube at 25°C, using a Zetasizer 3000HS (Malvern, UK).

Percent Drug Entrapment

The suspension of SLNs in PBS (pH 7.4) was centrifuged at 15,000 rpm for 30 min. The supernatant was analyzed spectrophotometrically for flurbiprofen content at 247 nm. From the concentration of the drug in the supernatant, the amount of drug adsorbed on the surface of particles was determined. Before any study, surface-adsorbed drug was removed completely by above procedure. Then SLNs were dissolved in minimum quantity of methanol and volume was made up with PBS (pH 7.4), and the drug was quantified spectrophotometrically at 247 nm against the blank solution (Cintra 10 UV-visible spectrophotometer).

Percent drug entrapment of gel-bearing SLN formulations was determined by dissolving gel in minimum amount of distilled water followed by addition of methanol to dissolve lipid. After dilution with PBS (pH 7.4), absorbance was recorded at 247 nm against the blank using Cintra 10 UV-visible spectrophotometer.

In Vitro Drug Release Studies

In vitro drug release from different formulations was determined using Franz diffusion cell in which the donor compartment contained the formulation while the receptor compartment was filled with PBS (pH 4.5). The sink condition was maintained by using 40% v/v PEG-400 in PBS in the receptor compartment, and the temperature was maintained at 37 ± 1°C with the help of a circulating water bath. Samples (1 ml) were withdrawn at appropriate time intervals and compensated with equal quantity of PBS (pH 4.5) containing 40% v/v PEG-400. Samples were filtered through Whatmann filter paper and drug content was determined spectrophotometrically at 247 nm (Cintra 10 UV-visible spectrophotometer).

In Vitro Drug Skin Permeation Studies

The in vitro drug skin permeation study of different formulations was performed using Franz diffusion cell through pig skin. The PBS (pH 7.4) containing 40% v/v PEG-400 was used as the receptor medium in the diffusion cell. The skin was sandwiched between the receptor compartment and donor compartment so that the dermal portion was continuously bathed with the receptor fluid maintained at 37 ± 1°C by circulating water bath and stratum corneum side exposed to ambient temperature. The content of the receptor fluid was stirred continuously using a magnetic stirrer. Samples were withdrawn at different time intervals, replaced with same volume of fresh solution, filtered, and amount of drug was determined spectrophotometrically at 247 nm (Cintra 10 UV-visible spectrophotometer).

In Vivo Performance Studies

Carrageenan-induced paw edema method was used to study the in vivo performance of the prepared drug delivery system, and the study was approved by University Animal Ethical Committee. Anti-inflammatory activity was determined by measuring change in the volume of inflamed paw, produced by injection of carageenan (0.1 ml of 1% w/v) using plethysmograph.

Male albino rats (Wistar strain) selected for the study were weighed and marks were made on the right hind paw just behind tibia-tarsal junction on each animal. Thus, every time the paw was dipped in the plethysmograph up to the fixed mark to ensure constant paw volume. Animals were divided into three groups including one controlled group with each group comprised of 6 animals. The formulations SLN-T4-gel, SLN-T4, and plain drug flurbiprofen solubilized in PBS (pH 7.4) in the dose of 3.8 mg/kg body weight was applied topically to albino rats of respective groups, excluding the animals of controlled group. The controlled group animals were injected with saline (0.9% NaCl) containing no drug. After 30 min of topical application of formulations, 0.1 ml of 1% w/v carrageenan (in 0.9% normal saline) was injected in the subplanter region of the right hind paw of rats. The initial reading just after injection and subsequent paw volumes was measured up to 8th hr with 1-hr interval and after 24 hr. The percent inhibition of edema induced by carrageenan was calculated for each group using the following equation: where, V_control = mean oedema volume of rats in controlled group and V_treated = edema volume of each rat in test group.

Statistical Analysis

Analysis of variance test (ANOVA) was applied to determine whether the results obtained from the experiment were significant or not. A probability level of p < .05 was considered to be significant.

RESULTS AND DISCUSSION

Solid lipid nanoparticles have been prepared from microemulsion technique by dispersing o/w microemulsion in a cold aqueous surfactant medium under mechanical stirring. Microemulsions are clear, thermodynamically stable, optically isotropic systems, obtained spontaneously by mixing surfactant, co-surfactant, oil, and water. The droplet structure is already contained in the microemulsion and thus their preparation does not require energy (Gaso 1997; Boltri et al. 1993). In the aqueous medium, SLN is formed by solidifying the oil droplets present in the o/w microemulsion. Consequently, the nanoparticle size is affected by the composition of the microemulsion system, particularly by the surfactant and co-surfactant used, as well as by the experimental parameters (Cavalli et al. 1996).

The process parameters involved in the preparation of SLN were optimized, including lipid:lipid ratio, lipid:surfactant ratio, and sonication time to obtain small nanoparticles with maximum drug entrapment. A drastic increase in particle size was observed by the use of stearic acid alone. The size of SLN decreased from 990 to 640 nm as on incorporation of cholesterol in increasing concentration (9:1 to 1:9 w/w ratio to stearic acid). The increase in particle size is related with an increase in particle agglomeration, which may be lowered by cholesterol incorporation (Table 1).

TABLE 1 Optimization of stearic acid/cholesterol ratio with regard to average particle size

	Ratio of	Average
Formulation	stearic acid:	particle	Polydispersity
code	cholesterol (w/w)	size (nm)	index
SLN-I	9:1	990	0.894
SLN-II	8:2	920	0.813
SLN-III	6:4	890	0.939
SLN-IV	5:5	880	0.652
SLN-V	4:6	720	0.417
SLN-VI	2:8	700	0.448
SLN-VII*	1:9	640	0.379

The soya lecithin concentration was optimized to obtain SLN selectively and avoid the formation of liposomes by CFquenching study. At the 0.2/1 w/w ratio of soya lecithin/lipid, the percent CF recovered was 89.35% (Table 2). Upon increasing the soya lecithin concentration upto 0.5/1 w/w of lipid, a slight variation in percent CF recovered (86.92%) was observed. However, a further change in soya lecithin concentration to 0.6/1 w/w of lipid, led to a drastic decrease in percent CF recovered (78.35%) due to formation of vesicular structure leading to quenching of CF. On further increase in soya lecithin quantity, a considerably higher reduction in the percent CF recovered (62.93%) was observed, which could be a consequence of increased liposome formation. These results indicate the optimal soya lecithin/lipid ratio (0.5/1 w/w) for maximal SLN formation.

TABLE 2 Optimization of soya lecithin/lipid ratio to maximize solid lipid nanoparticles/liposomes ratio

		Quantity of
	Ratio of soya	carboxyfluorescein
Formulation	lecithin/lipid	recovered
code	(w/w)	(%)
SLN-VII A	0.2/1	89.35
SLN-VII B	0.4/1	88.71
SLN-VII C*	0.5/1	86.92
SLN-VII D	0.6/1	78.35
SLN-VII E	0.8/1	69.37
SLN-VII F	1/1	62.93

The quantity of pluronic F-68 in the formulations was optimized to produce small nanoparticles with maximum percent drug entrapment. At the 0.4/1 w/w ratio of pluronic F-68 to lipid, a minimum average particle size of 620 nm and maximum percent drug entrapment 86.1% were recorded. Upon increasing the concentration of pluronic F-68 from 0.4/1 to 1/1 w/w ratio of lipid, an increase in average particle size (807 nm) was recorded, whereas the percent drug entrapment decreased considerably to 58.2% (Figure 1). The formation of some aggregates in the formulations may be the reason.

FIG. 1 Effect of pluronic F-68/lipid w/w ratio on average particle size and percent drug entrapment in SLN dispersions.

Sonication time also was optimized to achieve stable formulation with minimum average particle size and maximum percent drug entrapment. A stable SLN formulation was achieved after sonicating the formulation for 5 min with minimum average particle size and maximum percent drug entrapment, i.e., 560 nm and 91.7%, respectively. A further increase in sonication time (6 to 8 min) resulted in an increased average particle size (882 nm) and decreased percent drug entrapment (83.7%) (Figure 2). This may be due to the agglomeration of particles from generation of some surfacial charge.

FIG. 2 Effect of sonication time on average particle size and percent drug entrapment in SLN dispersions.

Various SLN dispersions bearing flurbiprofen (FB) were prepared using optimized concentration of stearic acid (100 mg), cholesterol (900 mg), soya lecithin (500 mg), pluronic F-68 (400 mg), butanol (2 ml), and drug (100 mg) with varying sonication times (2 to 5 min).

Shape and surface morphology of the prepared SLNs were evaluated by scanning electron microscopy. The study revealed that most of the SLNs was fairly spherical in shape and the surface of the particle showed a characteristic smoothness (Figure 3).

FIG. 3 Scanning electron photomicrograph of SLN.

Laser light scattering, the most powerful technique for the measurement of particle size, measures the fluctuation of the intensity of the scattered laser light, which is caused by particle movement. The polydispersity index is a measure of the distribution of nanoparticles (Westeren and Siekmann 1997). A laser particle size analyzer yields the diameter of the bulk population (average) and a polydispersity index to characterize the distribution ranging from 0.000 to 0.500 (Olbrich and Muller 1999). The polydispersity index greater than 0.500 showed the aggregation of particles. The combination of surfactants prevents particles agglomeration more efficiently and hence two surfactants pluronic F-68 and soya lecithin were selected for SLN preparation. The addition of co-emulsifying agent decreases the particle size, too. Butanol was added for this purpose.

The measurement of the zeta potential allows predictions about the storage stability of colloidal dispersion (Siekmann and Westesen 1996). In general, particle aggregation is less likely to occur for charged particles (high zeta potential) due to electric repulsion. Lower zeta potential facilitates aggregation. The formulation SLN-T4 showed high zeta potential value as compared with other formulations (Table 3).

TABLE 3 Zeta potential of solid lipid nanoparticles dispersions

Formulation	Zeta	Percent drug
code	potential (mV)	entrapment
SLN-T1	−20.2	71.50 ± 0.75
SLN-T2	−18.6	89.21 ± 0.42
SLN-T3	−17.1	78.89 ± 0.64
SLN-T4*	−49.4	92.70 ± 0.28
SLN-T1-Gel	—	67.70 ± 0.80
SLN-T2-Gel	—	83.70 ± 0.32
SLN-T3-Gel	—	72.22 ± 0.55
SLN-T4-Gel*	—	89.98 ± 0.74

In SLN dispersions the percent drug entrapment was found to be 71.5, 89.21, 78.89, and 92.7% for SLN-T1, SLN-T2, SLN-T3, and SLN-T4, respectively. The same SLN dispersions incorporated in gel showed the percent drug entrapment to be a little bit lower, which may be due to distribution of SLN dispersion in gel (Table 3).

The prepared formulations were first studied to establish the release kinetics of the drug from the SLN systems. To evaluate the release pattern, the scientifically recommended Franz diffusion cell was chosen. The formulation was placed in the donar compartment of Franz diffusion cell. The PBS (pH 4.5) containing 40% v/v PEG-400 was used as diffusion medium because the pH of the skin (stratum, corneum) is relatively acidic (pH 4.5–5.5) compared with the pH of the dermal side (Chatterjee 1966). A different release kinetic was observed for the SLN dispersions and SLN-gel formulations. Fick's law of diffusion seems not to be applicable in each case. An initial rapid drug release was noted in the SLN dispersions, whereas a lag time (∼15 min) was observed with SLN-gel formulations which could result from the time taken by the drug to diffuse across the gel. The direct exposure of SLN dispersion to diffusion media and quick release of drug may account for rapid initial release in SLN dispersions. Both SLN dispersions and SLN-gel formulations showed controlled drug release over 6 hr and an increase in release rate was observed after 24 hr.

The log percent cumulative drug released was plotted as a function of log time and the slope of the curves was determined as the values of diffusional release exponent (η) (insets in Figures 4 and 5). The values of diffusional release exponent (η) from the straight lines were noted to be 0.7–0.8 in SLN dispersions and SLN-gel formulations, which showed that the release of drug from formulations followed a non-Fickian pattern (Langer and

FIG. 4 In vitro drug release profile of SLN dispersions in PBS (pH 4.5).

FIG. 5 In vitro drug release profile of SLN-gel formulations in PBS (pH 4.5).

Peppas 1981). From the percent cumulative drug released versus time^0.8 plot, the slope values were determined as release rate constants (Figures 4 and 5). The percent cumulative drug released was maximum for the products SLN-T1 (85.43%) and SLN-T1-gel (75.98%) with release rate constants of 6.8647 and 8.5797%/cm²/hr^0.8, respectively. SLN-T4 (64.13%) and SLN-T4-gel (61.93%) have minimum drug release with release rate constants of 5.2526 and 6.9102%/cm²/hr^0.8, respectively (Table 4). Hence, SLN-T4 and SLN-T4-gel showed better results than other formulations. SLN-T4-gel slowly releases the drug as compared with SLN-T4 dispersion, accounted for by the time the drug takes to diffuse through gel. The slower release of drug from SLN-T4-gel maintained the drug concentration for longer period of time. Burst releases as well as sustained release both are of interest for dermal application. Burst release can be useful to improve the penetration of drug. Sustained release supplied the drug over a prolonged period of time.

TABLE 4 Rate constants of in vitro drug release and in vitro drug skin permeation of various formulations

Formulation	Release rate constant	Skin permeation rate
code	(Percent/cm^2/hr^0.8)	constant (Percent/cm^2/hr)
SLN-T1	6.8647	8.4188
SLN-T2	6.3282	7.3612
SLN-T3	6.4527	7.8233
SLN-T4	5.2526	5.207
SLN-T1-Gel	8.5797	8.7815
SLN-T2-Gel	7.9098	7.9843
SLN-T3-Gel	8.1112	8.3893
SLN-T4-Gel	6.9102	5.2243

The in vitro drug permeation through pig skin was observed for different formulations using Franz diffusion cell. The release kinetics was established by determining the diffusional release exponent from the plot of log of cumulative drug permeated versus log time. This plot yielded a straight line from which diffusional release exponent (η) was calculated and found very near to 1.0 for both SLN dispersions and SLN-gel formulations (Langer and Peppas 1981) (Figures 6 and 7). In this way, we concluded that the release of drug from formulations followed zero-order kinetics. A lag time (15–30 min) was observed in every case but more in SLN-gel formulations because in these formulations the drug has to cross two diffusion barriers, one the gel and the other is skin (insets in Figures 6 and 7).

FIG. 6 In vitro drug skin permeation profile of SLN dispersion in PBS (pH 7.4).

FIG. 7 In vitro drug skin permeation profile of SLN-gel formulations in PBS (pH 7.4).

From the percent cumulative drug permeated versus time plot, the slope values were determined as the skin permeation rate. The skin permeation rate constants were found to be 5.207 and 5.2243%/cm²/hr in SLN-T4 and SLN-T4-gel, respectively (Table 4). Within 24 hr, the fluid SLN dispersion turned slowly into a semisolid gel. Gel formation of SLNs can be correlated with polymorphic transitions of the lipid matrix. Because of a change in the form of the lipid lattice, drug expulsion occurs as a consequence of this transition and sustained release properties of the carrier system are lost (Freitas and Muller 1999). The expelled FB is very poorly soluble in water and hence thermodynamic activity of drug increases. The increase in thermodynamic activity can explain the higher diffusion velocity. For pure SLN dispersion, water loss and induced polymorphic transitions were a likely key to the release profile. In SLN-gel, water evaporation is reduced because of water-binding properties of glycerol and the polymer polyacrylamide. Therefore, polymorphic transitions and successive drug expulsion should be reduced.

Once more the SLNs performed a polymorphic transformation with subsequent drug expulsion. However, this transformation was slower in SLN-gel compared with fluid SLN dispersion (Jenning, Shafer-Korting, and Gohla 2000). Under the chosen experimental conditions the SLN dispersion and SLN-gel formulations possessed a sustained drug release over 24-hr period. But this sustained effect was more pronounced with SLN-gel formulations.

The in vivo performance of selected SLN-T4 dispersion and SLN-T4-gel were carried out in carrageenan-induced rat paw edema model. The formulation under study not only decreased the inflammation to the larger magnitude, but also sustained this magnitude. In SLN-T4-gel the maximum inhibition was observed at 5-hr with higher value (83%), upto 8-hr inhibition was maintained above 55%, and even after 24-hr, 30% inhibition was observed. However, in case of plain drug maximum inhibition was displayed at 3-hr with magnitude of 63% and just after 4-hr it scored below 50% (Figure 8). The possible reason could be the drug concentration in the blood, which was maintained for longer duration in case of SLN-T4-gel in comparison to plain drug. In comparison to these two formulations SLN-T4 gives intermediate results. The maximum inhibition for SLN-T4 was observed at 3-hr and upto 8-hr inhibition was maintained and even after 24-hr inhibition was observed (Figure 8). The lower anti-inflammatory activity of SLN-T4 as compared with SLN-T4-gel is due to slower release of drug from SLN-T4-gel, which maintained for longer period of time. This was attributed to gel structure and the surface-active properties of the gel.

FIG. 8 Anti-inflammatory activity of different formulations in carrageenan-induced rat paw edema.