Formulation and Evaluation of Nanoparticles of Ketoprofen for Transdermal Delivery

INTRODUCTION

Transdermal drug delivery system is the novel drug delivery system that breaks many barriers like need of assistance, uncomfortable administration and intermediate dosing.¹ For local and systemic delivery of drugs, transdermal route of administration is one of the most potential routes of drug administration,² as the skin is an effective medium from which absorption of the drug takes place and enters into systemic circulation over a period of time.³ Nanoparticles (NPs) are the simplest form of structures with size less than 100nm.² The advantages of nanoparticles as a drug delivery system are - no wastage of drugs thus enhanced bioavailability of drug at specific site in right proportion for a prolonged period of time,improved solubility of poorly water soluble drugs, prolonged half-life of drug systemic circulation by reducing immunogenicity, release drug at sustained rate and lower the frequency of administration⁴ . Various method of preparation of nanoparticles are solvent evaporation, emulsification, nano-precipitation, ionic gelation etc.⁵

The primary mechanism of action for ketoprofen is considered to be inhibition of the cyclooxygenase pathway of arachidonic acid metabolism, leading to decreased production of prostaglandins. Based on in vitro experiments, ketoprofen is considered as one of the more potent inhibitors of prostaglandin synthesis. It appears to be a relatively nonselective inhibitor of cyclooxygenase isozymes. Inhibition of cyclooxygenase prevents the formation of these prostaglandins and the inflammation they cause. The antipyretic effect is seen to occur by inhibition of prostaglandins in the hypothalamus and analgesic effect is by the interruption of mediators of inflammation, such as bradykinin, and prevention of their effect on peripheral pain endings. Ketoprofen can cause gastrointestinal irritation that may lead to ulceration which can be overcome by transdermal delivery of ketoprofen.⁴

MATERIALS AND METHODS

 Materials

Ketoprofen was purchased from BMR Pharma and Chemicals, Hyderabad. HPLC grade solvents were purchased from Ranbaxy Fine Chemicals Ltd., New Delhi. Phosphoric acid was purchased from Karnataka Fine Chem. Double distilled water was prepared in laboratory. Porcine ear tissue obtained from local slaughter house.

Methods

Preparation of Ketoprofen loaded Oleic acid nanoparticles:^7,8

 Microemulsification and solvent evaporation method was used for the preparation of nanoparticles. The method includes various lipids at different amounts and various amount of drug.6 To prepare oil phase- drug, oleic acid & Span60 were dissolved in ethanol with the help of bath sonicator for 45 minutes. Then to prepare aqueous phase- Tween 80 and water were dissolved in another beaker with help of magnetic stirrer. Oil phase was slowly injected into the aqueous phase with the help of 5ml syringe fitted with 23 - gauze needle and stirred over a magnetic stirrer for five hours to evaporate ethanol completely. To get uniform size of nanoparticles and for proper mixing, it was homogenized for 5 minutes at 9000 rpm (IKA T-25 ULTRA-TURRAX Digital High[1]Speed Homogenizer Systems). The sample was taken and the particle size was checked using nanosizer of HORIBA Scientific (Nano Partica) – SZ 100. Similarly O5 to O8 oil phase was prepared using acetone in place of ethanol using the same method of preparation.

Preparation of Ketoprofen loaded GMS nanoparticles:

 Using the same method as discussed in above section, nanoparticles of Ketoprofen were formulated using GMS in place of Oleic acid (G1 – G8).

Preparation of Ketoprofen loaded Cholesterol nanoparticles:

Nanoparticles of Ketoprofen were formulated using Cholesterol in place of Oleic acid (C1 –C8). The composition of nanoparticles using oleic acid, using GMS, using cholesterol are shown in Table 1, Table 2 and Table 3 respectively.

Evaluation of Nanoparticles

After the preparation of nanoparticles of Ketoprofen for transdermal delivery, various evaluation tests were carried out as mentioned below.

Drug content:

1 ml of the nanoparticles dispersion was dissolved in ethanol and the sample was diluted appropriately with pH 7.4 phosphate buffer to get the concentration in the standard graph range and drug content was estimated by UV-Spectrophotometry at 260nm.

 Zeta-Potential determination:

The prepared SLN sample was diluted (1 ml into 5 ml) using double distilled water. Then, the samples were transferred into the zeta cuvette of carbon electrodes and kept inside the instrument. Then, the laser scattering light was passed through cuvette tube and analyzed the zeta potential using Horiba Scientific (Nano Partica) – SZ100.

Particle size analysis and Poly dispersity index:

 The size of prepared nanoparticles played a vital role for giving the better absorption and bio availability. For nanoparticles, the particle size must be in the range of 10nm - 100nm. The formulated nanoparticles were diluted with double distilled water and the sample was transferred to cuvette and kept inside the instrument. Then, the laser scattering light was passed through cuvette tube and the particle size distribution was analyzed by using Horiba Scientific (Nano Partica)- SZ100.

The polydispersity is characterized as particles of varied sizes in the dispersed phase of a disperse system compared mono-disperse systems. Polydispersity Index is dimensionless and scaled such that values smaller than 0.05 are rarely seen other than with highly mono-disperse standards. Values greater than 0.7 indicate that the sample has a very broad size distribution and is probably not suitable for the Dynamic Light Scattering (DLS) technique. The various size distribution algorithms work with data that falls between these two extremes. The polydispersity index (PDI) must be smaller than 0.6-0.7 to have a reliable measurement.⁹

Diffusion Study of Ketoprofen from NPs:

The Franz diffusion cells were thoroughly cleaned with distilled water, the porcine ear tissue of freshly slaughtered pigs was taken and hair was removed. The epidermal skin was surgically separated using micro-dissecting scissors and blunt forceps, then it was dipped into hot water (600 ) to remove subcutaneous fat followed by washing with water and the prepared porcine ear epithelium was placed between the donor and receptor compartments of the diffusion cell, the permeation studies were initiated within 1hour of isolating ear epithelium. The pH 7.4 phosphate buffer was filled into the receptor compartment and air bubbles were totally removed from the receptor compartment. O2 formulation of Ketoprofen NPs equivalent to 10mg of Ketoprofen was added into the donor compartment. Then the entire set up was placed over magnetic stirrer and temperature was maintained at 370 by placing the diffusion cell in a water bath for 24 hours at 370 . Then 1ml of samples from the bottom portion of receptor compartment were withdrawn at 1hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs 12 hrs, 17 hrs and 24 hrs and replaced with 1 ml of pH 7.4 phosphate buffer. Amount of drug permeated at each sample was estimated using HPLC method.⁹ Amount of drug permeated per sq.cm at each time interval was calculated and flux was calculated from slope of the line plotted by taking amount permeated per sq.cm Vs time. Permeation of drug from nanoparticles was compared with permeation of drug from 5mg and 10mg of drug solution.⁹

RESULTS

DISCUSSION

Evaluation of Nanoparticles of Ketoprofen:

Nanoparticles were evaluated for particle size, drug content, zeta potential, and polydispersity index. All the formulations were prepared using S/CoS ratio 1:8 of Span 60 and Tween 80 and 10, 20, 30, 40 mg of Ketoprofen using 100mg of lipids such as Oleic acid, GMS, Cholesterol respectively. Trials were also taken to increase the amount of lipids (more than 100 mg) and amount of drug (more than 40 mg) using the same S/CoS ratio. But ethanol could not dissolve the increased amount, but acetone could dissolve. But upon estimation of particle size, it was found that the polydispersity index was more and multiple dispersions peaks were observed in particle size estimation. So the study was limited to 100 mg of lipids and upto 40 mg of drug addition. The results of all evaluation parameters were shown in Table 4.

Drug Content

The drug content of formulation O1 to O8 (Oleic acid), formulation G1 to G8 (GMS), and Formulation C1 to C8 (Cholesterol) were shown in the Table 4. Among all the formulations, oleic acid formulation have shown more drug content. Among oleic acid formulations, O2 was found to have comparatively highest drug content (96.14%).

 Zeta Potential

Zeta potential of formulations O1 to O8 (Oleic acid) were found to be -10.6mV to -16.8mV. Formulations G1 to G8 (GMS) were found to possess -7.8mV to -23.1mV zeta potential. Formulations C1 to C9 (Cholesterol) were found to possess -15.4mV to -30.9mV zeta potential. The zeta potential for moderate stability should be in the specified range between ±30mV to ±40mV.10 So, out of twenty four formulations O2 showed better zeta potential (-12.8 mV) which maintained the optimum range for transdermal delivery of nanoparticles.

 Particle Size Distribution and Polydispersity Index Particle size distribution and polydispersity index of formulations O1 to O8 (Oleic acid) were found to be 66.8nm to 278.1nm and 0.283 to 0.857 respectively. Formulation G1 to G8 (GMS) were found 146.5nm to 409.9nm and 0.196 to 0.857 particle size distribution and polydispersity index respectively. Formulation C1 to C8 (Cholesterol) were found to have 187.8nm to 299.1nm and 0.241 to 0.878 particle size distribution and polydispersity index respectively as shown in the Table 4. So, out of twenty four formulations O2 showed smaller particle size distribution and polydispersity index.

Particle size was observed to increase with increase in the amount of lipid in all the three lipid preparations. When the solvent for oil phase was changed from ethanol to acetone,particle size was found to increase and PDI was also comparatively more (broader particle size distribution peak) than with ethanol as solvent.

Similarly, when solvent acetone was used, drug content was also observed to be less in comparison to ethanol. Among all the three lipids, NPs prepared from Oleic acid has smaller size, next size was observed with GMS NPs, the larger among the three was Cholesterol NPs. As the carbon number is increased in the lipid then the particle size is also found to increase at a constant ratio of S/CoS. The molecular formula of Oleic acid is (C18H34O2), for GMS (C21H42O4) and Cholesterol is (C27H46O). Based on particle size and drug content, O2 was selected as best formulation which has particle size 66.8nm and drug content was 96.14%. Particle size distribution of O2 formulation was shown in Figure 1. Further study was carried out on O2 formulation for ex-vivo transdermal permeation study.The flux (J) was observed when 5ml of prepared nanoparticles equivalent to 2mg/ml of Ketoprofen was placed into donor was 0.0031 µg/ sq cm/hr. The flux (J) observed when drug solution of 5mg,10mg and nanoparticles equivalent to 10 mg was found to be 0.0002 µg/sq cm/hr, 0.0006 µg/sq cm/hr and 0.0031µg/sq cm/hr respectively, which indicates that nanoparticles preparation has superior permeability compared to pure drug solutions.¹¹ The increase in flux indicates the nanoparticles formulation of Ketoprofen for transdermal delivery is successful. The observed increase in permeability could be due to the smaller size of the particles as well as S/CoS, used in the preparation which also act as penetration enhancers. On the whole due to less particle and due to the effect of surfactant, flux was increased 5 times when compared with equivalent drug solutions (Figure 2).

CONCLUSSION

 Ketoprofen loaded Nanoparticles were prepared using oleic acid, GMS and Cholesterol for Transdermal delivery. Lipid nanoparticles were formulated using oleic acid, GMS and cholesterol and Tween 80 and Span 60 were used as co-surfactant and surfactant. Twenty four (24) different formulations of Ketoprofen loaded oleic acid, GMS and Cholesterol nanoparticles were prepared by Micro-emulsification Method and Solvent Evaporation Method.

Formulations were planned using 10, 20, 30 and 40 mg of drug using S/CoS ratio 1:8. The prepared nanoparticles were evaluated for Drug content, Particle size, Poly-dispersity index, Zeta-potential and ex-vivo transdermal permeation study.¹²

Among all the formulations, O2 was found to have comparatively highest drug content (96.14%) with less particle size was selected as the optimum formulation and subjected for ex vivo transdermal permeation study. The flux for nanoparticle diffusion study was found to be 0.0031 μg/sq cm/hr, which indicates the superior permeation characteristics compared to pure drug solution.