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Solid Lipid Nanoparticles of Clobetasol for Psoriasis

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Formulation and Evaluation of Solid Lipid Nanoparticles of Clobetasol for Topical Treatment of Psoriasis

CHAPTER 4 Methodology

4. METHODOLOGY

MATERIALS USED:

Table 5: LIST OF CHEMICALS USED WITH SUPPLIERS

MATERIALS

NAME

SUPPLIERS

Drug

Clobetasol

Dr Reddy’s labs, Hyderabad

Excipients

Carbopol 934

Loba Chemie Pvt. Ltd, Mumbai

Stearic acid

Loba Chemie Pvt. Ltd, Mumbai

Bees wax

Loba Chemie Pvt. Ltd, Mumbai

Span 20

Loba Chemie Pvt. Ltd, Mumbai

Tween 20

Loba Chemie Pvt. Ltd, Mumbai

Chemicals

Sodium Hydroxide

Ranbaxy Fine Chemicals Ltd, Mumbai.

Methanol

Loba Chemie Pvt. Ltd, Mumbai

Potassium dihydrogen

ortho phosphate

Thermo Electron LLs India Pvt. Ltd

Table 6: LIST OF EUIPMENTS USED WITH SUPPLIERS

SL. NO.

INSTRUMENT

MANUFACTURE

1

Electronic balance (BL-220H)

Shimadzu Corporation, Japan

2

UV/VIS Spectrophotometer UV 2301

Shimadzu Corporation, Japan

3

Polytron PT 1600E

Kinematica AG, Switzerland.

4

Particle size analyzer

Malvern, U.K

5

Zeta potential instrument

Malvern, U.K

6

FTIR Spectrophotometer

Shimadzu 8400 series

7

Scanning Electron Microscopy

Model JSM 840 A, Jeol, Japan

8

pH meter

Digisun Electronics, Mumbai

9

Magnetic stirrer

Remi Eqipments, Mumbai.

10

Brookfield viscometer

Brookfield engineering

Laboratories. USA

11

Franz diffusion cell

--

Physicochemical study on the drug:

  • Melting point determination

Melting point is the temperature at which the pure liquid and solid exist in equilibrium. In practice it is taken as equilibrium mixture at an external pressure of 1 atmosphere; this is sometimes known as normal melting point. The Thiel’s tube method of melting point determination liquid paraffin was used in present study.

  • UV spectrum

UV scanning was done for pure drug from 200-400 nm in the dilution medium of methanol and in the dilution medium of phosphate buffer pH 7.4

Dilution medium

λmax

Methanol

240nm

Phosphate buffer

pH 7.4 solution

239nm

Standard calibration curves of Clobetasol:

  • Reagents
  • Methanol
  • Phosphate Buffer pH 7.4 114,115
  • Medium- Methanol 116

50 mg accurately weighed CP was dissolved in the methanol and volume was made up to 50ml with methanol [Stock 1]. From stock 1, different dilutions were prepared in the concentration range of 5, 10, 15, 20 and 25µ g/ml using methanol as dilution medium. The absorbance of these solutions was measured against blank as methanol in UV spectrophotometer at 240 nm.

  • Medium- Phosphate buffer pH 7.4 solution

50 mg accurately weighed CP was dissolved in the methanol and volume was made up to 100ml with methanol [Stock 1]. From stock 1, different dilutions were prepared in the concentration range of 5, 10, 15, 20,

25, 30µ g/ml using Phosphate buffer pH 7.4 solution. The absorbance of these solutions was measured against Phosphate buffer pH 7.4 solution as blank in UV spectrophotometer at 239 nm.

Compatibility studies of drug and polymers:

FTIR spectra of pure clobetasol, physical mixtures, and SLN formulations are carried out to determine if there was any interaction between the drug and the other formulation components117.

Since IR is related to covalent bonds, the spectra can provide detailed information about the structure of molecular compounds. In order to establish this point, comparisons were made between the spectrum of the substances and pure compound.

The Preformulation study was carried out prior to the development of the dosage forms. The compatibility of the drug and the excipients used were determined by IR spectrometer Shimadzu 8400 series. The spectra of formulations were compared with that of pure drug in order to ascertain for any possible interaction between polymer and drug.

Preliminary studies:

Initially, SLN was prepared by solvent injection method by using clobetasol (drug), carnauba wax and beeswax as lipid phase, cetyl alcohol and lecithin soya as surfactants. Tween 20 as co surfactant phase and finally distilled water to make up the volume. Where in this method, both liquid phase and lipid phase were heated to 70-75oC. When lipid phase was heated to desired temperature drug was dispersed in it and added to aqueous phase under the magnetic stirrer for 30mins. The lipid phase is added in to aquoes phase by drop wise using syringe. Thus SLNs were formed due to rapid crystallization of oil droplets and precipitation. This respective formulation design is shown in table 7. But the formed SLN was not dried and unstable. Therefore lipid extrusion method was used to prepare various formulations with different concentrations of lipid in an effort to optimize the formulations for the particle size ranging in nano scale.

Table 7: Formulation design for solvent injection method

Ingredients

Quantity (gm)

Clobetasol

25mg

Carnauba wax

1.25

Beeswax

1.25

Tween 20

1.87

Lecithin soya

1.87

Cetyl alcohol

1.25

Distilled water q.s to

50

The lipid extrusion method was adopted to prepare SLN.

  • At 25mg of CP is kept constant in all formulation.
  • Initially, the drug with different lipids tried with different concentration constant speed. The SLN were evaluated for particle size.
  • The composition of formulation is shown in table.

Formulation design:11,17,18

Procedure for preparation of SLN by lipid extrusion technique:

  • Lipid extrusion is carried out at temperatures above the melting point of the lipid and is similar to the homogenization of an emulsion.
  • A pre-emulsion of the drug loaded lipid melt and the aqueous emulsifier phase (same temperature) is obtained by high-shear mixing device (like Polytron PT 1600E homogenizer).
  • High pressure homogenization of the pre-emulsion is done above the lipid melting point. Usually, lower particle sizes are obtained at higher processing temperatures because of lowered viscosity of the lipid phase.

Fig 11: Schematic representation of SLN preparation by lipid extrusion

Table 8: Formulation design for lipid extrusion method

Ingredients

F1

F2

F3

F4

F5

F6

Clobetasol

25

25

25

25

25

25

Bees wax

500

600

800

1000

1500

2000

Carnauba wax

250

300

600

1000

1500

2000

Cetyl alcohol

250

300

400

600

800

800

Lecithin soya

200

200

200

200

200

200

Tween 20

250

250

250

250

250

250

Carbopol 934

500

500

500

500

500

500

Distill water QS

50

50

50

50

50

50

Table 9: Formulation design with stirring speed and duration of rotation using Polytron pt 1600E for Formulation F

Formulation codes

Stirring speed(RPM)

Duration of rotation

(Minutes)

F1

15000

30

F2

15000

30

F3

15000

30

F4

15000

30

F5

15000

30

F6

15000

30

EVALUTION OF SLN: Physical Evaluations:

Visual appearance

pH:

The pH of SLN formulations were measured using pH paper.

Rheological studies

Rheological properties (study of deformation and flow of matter) are required in various pharmaceutical areas. It helps to monitor the effect of vehicles consistency on release of drug from the preparations and subsequent percutaneous absorption. Also it is important from the manufacturing point of view. Viscosity measurements were carried out using a Brookfield viscometer (T – bar spindle). The formulation of SLN was kept in 100ml beaker and dial readings was noted at 3, 5, 6, 10, 12, 20, 30, 50 and 60 rpm. The speed was then successively lowered and the corresponding dial readings were noted.

Particle Size Analysis118:

The particle size should be less than 1000 nm in nanoparticles. It can be analyzed by using Malvern particle size analyzer. Particles in the size range of colloids display constant random thermal motion which is known as Brownian motion. This motion causes the intensity of light scattered by the particles to vary with time. The larger the particle slower their motion and hence the smaller the variation in intensity of light scattered. Photon correlation spectroscopy uses the rate of change in the intensity to determine the size distribution of particles. The zetasizer has a correlator with 64 channels. Each of this channel measures changes in light

fluctuation over a defined time span. The time span is known as the sample time or delay time the correlator measures the light intensity by counting photons. For a very short time period the changes in light intensities will be very small as the particles had very little time to move. The position of the particles can be statistically defined as being highly correlated, Contrast to this with a long sample time, particles will have moved randomly from their initial positions. Therefore the particles can be described statistically as not being correlated.

Zeta potential measurement

Zeta potential of the SLNs were measured by malveren zetasizer. The zetasizer mainly consist of laser which is used to provide a light source to illuminate the particles within the sample. For zetapotential measurements this light splits to provide an incident and reference beam. The incident laser beam passes through the centre of the sample cell, and the scattered light at an angle of about 130 is detected. when an electric field is applied to the cell, any particles moving through the measurement volume will cause the intensity of light detected to fluctuate with a frequency proportional to the particle speed and this information is passed to the digital signal possessor and then to a computer. Zetasizer software produces a frequency spectrum from which the electrophoretic mobility hence the zeta potential is calculated.

Scanning Electron Microscopy (SEM):

Surface morphology of the specimen will be determined by using a scanning electron microscope.

Procedure:

The samples are dried thoroughly in vaccum desicator before mounting on brass specimen studies, using double sided adhesive tape. Gold-palladium alloy of

120°A Knees was coated on the sample sputter coating unit (Model E5 100 Polaron U.K) in Argon at ambient of 8-10 with plasma voltage about 20mA. The sputtering was done for nearly 5 minutes to obtain uniform coating on the sample to enable good quality SEM images. The SEM was operated at low accelerating voltage of about 15KV with load current about 80mA.The condenser lens position was maintained betwee 4.4-5.1. The objective lens aperture has a diameter of 240 microns and working distance WD=39mm.

Drug content119:

The drug equivalent to 10 mg of formulation was taken and dissolved in small quantity of methanol. Then the formulation is warmed on the water bath so that the drug present in the formulation was completely dissolved. Then the solution was filtered through Whattman filter paper in 25 ml. volumetric flask and volume was made up to the mark by methanol to give concentration of 1000 μg/ml. for Clobetasol. Then 1 ml. was pipetted out in 100 ml. volumetric flask to give concentration of 10μg/ml and then absorbance was measured at 240 nm.

In-vitro release studies114,115,120:

In Franz diffusion cell, 6 gm. of sample was kept in donor compartment. The entire surface of cellophane membrane was in contact with the receptor compartment containing 50 ml of phosphate buffer pH 7.4. The receptor compartment was continuously stirred using the magnetic stirrer. The temperature was maintained 35°C. The study was carried out for 24 hrs and the sample was withdrawn at 30 minute time interval and same volume was replaced with free phosphate buffer. The content of clobetasol from withdrawn sample was measured after suitable dilution.

Stability studies:

Whenever a new formulation is developed, it is very essential to establish that the therapeutic activity of the drug has not undergone any change. To conform this, the selected formulations were subjected to stability studies. Generally, the observation of the rate at which the product degrades under normal room temperature requires long time. To avoid this undesirable delay, the principles of the accelerated stability studies are adopted.

The International Conference of Harmonization guidelines titled “stability testing for drug substance and product” describes the stability tests requirements for drug registration applications in the European Union, Japan and United States of America.

Table 10: International climatic zones and climatic conditions

Climatic

Conditions

Zone I

temperate

Zone II Mediterranean (su tropical)

Zone III Hot/dry or Hot/moderate RH

Zone IV Very hot/humid

Mean annual

temperature

20o C

21.6oC

26.4oC

26.7oC

Mean kinetic

temperature

20oC

22oC

27.9oC

27.4oC

Mean annual

relative humidity

42%

52%

37%

76%

Derived storage

conditions

21oC/45% RH

25o C/60% RH

30oC/35% RH

30oC/70% RH

Table 11: General stability testing consideration

Study

Storage condition

Minimum time period

covered by data submission

Long term

25°C ± 2°C/60% RH ± 5% RH

or

30°C ± 2°C/65% RH ± 5% RH

12 months

Intermediate

30°C ± 2°C/65% RH ± 5% RH

6 months

Accelerated

40°C ± 2°C/75% RH ± 5% RH

6 months

Table 12: General stability testing consideration

Study

Storage condition

Minimum time period covered by data submission

Long term

5 oC ± 3 oC

12 months

Accelerated

25oC± 2oC/60% RH ± 5%

RH

6 months

Stability studies were carried out at 40°C ± 2°C /75% RH ± 5% RH for the selected formulations for physical and chemical stability.


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