Formulation and Evaluation of Cubosomal Gel of an Anti-inflammatory Agent

 

Dr. Beena P¹*, Shithin Ann Varghese², Jinny Ann Alexander², Steffy P Raju², Sherin Koshy², Feba Elsa Mathew², Dr. Elessy Abraham³

¹Professor, Nazareth College of Pharmacy, Othera P.O Thiruvalla, Kerala, India - 689546.

²M. Pharm Delegate, Nazareth College of Pharmacy, Othera P.O Thiruvalla, Kerala, India - 689546.

³Principal, Nazareth College of Pharmacy, Othera P.O Thiruvalla, Kerala, India - 689546.

 

ABSTRACT:

The present research has been undertaken with the aim to develop a transdermal Cubosomal gel formulation of Ketoprofen, which would attenuate the gastrointestinal toxicities associated with oral administration. This research also aimed to encapsulate high drug pay load in Cubosomes for improved therapeutic efficiency. Cubosomes were prepared by Top- down technique. Different formulations (F1 – F9) were prepared and optimized for better performance in terms of drug content, SEM analysis, Zeta potential, entrapment efficiency and drug release. From F1 – F9 formulations, studies showed that F7 is better. Then it is formulated into gel using carbopol as gel base. The physical parameters like appearance, pH, viscosity, spredability, extrudability, ex- vivo drug release and in vitro skin irritation test using HET- CAM, were also evaluated. The cubosomal gel formulation (F7) was found to be clear without any aggregate indicating excellent homogenicity. The pH of the formulation was found close to neutral, indicating the absence of skin irritation. In vitro skin irritation study also reveals there is no skin irritation. The ex-vivo drug release study shows that the formulation (F7) has a good release rate when compared to other topical gels (87.2%). The kinetic study of the optimized formulation (F7) was also carried out and found that the formulation undergo zero order kinetics. The mechanism of drug release was found to be Higuchi model.

 

 

 

 

 

1.      INTRODUCTION:

Cubosomes are discrete, sub-micron, nanostructured particles of the bicontinuous cubic liquid crystalline phases. They consist of honeycombed structures separating two internal aqueous channels along with a large interfacial area. They contain similar microstructure as that of the parent with high surface area and their dispersions are less viscous than the parent cubic phases^[1]. Almost all cubosomes are composed of polymers, lipids and a surfactant with polar and non-polar components (amphiphilic). Due to the hydrophobic effect, amphiphilic molecules are driven into the polar solvent to impulsively identify and assemble into a liquid crystalline dispersion of nanometer scale. Thus cubosomes are bicontinuous cubic liquid phase enclosing two distinct regions of water, divided by surfactant controlled bilayers^[2].

 

 

1.1 Cubosomal Gel:

Cubosomal gels are the cubosomal dispersion of hydrogel. The term “Gel” was introduced in the late 1800 to name some semisolid material according to pharmacological, rather than molecular criteria. The U.S.P. defines gels as a semisolid system consisting of dispersion made up of either small inorganic particle or large organic molecule enclosing and interpenetrated by liquid. The inorganic particles form a three-dimensional “house of cards” structure. Gels consist of two-phase system in which inorganic particles are not dissolved but merely dispersed throughout the continuous phase and large organic particles are dissolved in the continuous phase, randomly coiled in the flexible chains^[3].

 

2. MATERIALS AND METHODS:

2.1 Materials:

Poloxamer-407, Glycerol mono oleate and Ketoprofen were purchased from Yarrow chemicals, Mumbai. Carbopol, Propylene glycol and Triethanol amine were purchased from Spectrum chemicals, Kochi. Carbopol, propylene glycol and triethanol amine were of commercial grade. All other reagents used were of analytical grade.

 

2.2 Methods:

2.2.1 Formulation of Cubosomal Dispersion:

Cubosomal dispersions of Ketoprofen were prepared by top-down technique. Accurately weighted quantity of Glyceryl monooleate (GMO) and poloxamer 407 polymer mixed and melted in a water bath at 60⁰C, to this mixture add Ketoprofen drug and stir until completely dissolved, then to this solution add drop by drop preheated (up to 70⁰C) distilled water of suitable quantity by continuous stirring for 2 hours, This whole system is taken into subjected for homogenization at 1500rpm for 1 minute under at room temperature. Thus formed liquid dispersion of cubosomes was kept at a room temperature, avoids direct sunlight and which will used for further study.^[4] The formulation design was given in table 1.

 

Table 1: Formulation Design of Cubosomes

Formulation	Poloxamer-407 [gm]	Glycerol monooleate [gm]	Ketoprofen [mg]	Water [ml]
F1	0.3	2.5	100	50
F2	0.3	2.0	100	50
F3	0.3	1.5	100	50
F4	0.25	2.5	100	50
F5	0.25	2.0	100	50
F6	0.25	1.5	100	50
F7	0.2	2.5	100	50
F8	0.2	2.0	100	50
F9	0.2	1.5	100	50

 

 

2.2.2 Evaluation of Cubosomal Dispersions:

2.2.2.1 Analytical Method Used in the Determination of Ketoprofen:

a) Determination of λmax of Ketoprofen:

Absorption maximum of pure Ketoprofen was determined by dissolving Ketoprofen in phosphate buffer saline pH 7.4. A sample of 10µg/ml was prepared and scanned for maximum absorbance using UV Visible spectrophotometer in the range from 200 - 400nm using phosphate buffer saline pH 7.4 as blank^[5].

 

b) Preparation of calibration curve of Ketoprofen:

10mg of ketoprofen was accurately weighed and transferred into 100ml volumetric flask. The drug was dissolved and made up to the volume with phosphate buffer saline pH 7.4. It was further diluted with same buffer to get concentration of 2, 4, 6, 8 and 10µg/ml. The absorbance of solution was measured spectrophotometrically at 259 nm using buffer as blank. The absorbance values were plotted against concentration to obtain the standard graph.

 

c) Compatibility studies:

Compatibility studies were done using FTIR and DSC.^[6,7]

 

2.2.2.2 Optical Microscopy

The cubosomal dispersions prepared were observed under binocular compound microscope at 10X and 40X magnification for studying the shape and surface morphology.^[5]

 

2.2.2.3 Particle Size and Polydispersity Index:

The mean particle size and particle size distribution was determined by Malvern nano Zeta sizer instrument. The vesicles after diluted with distilled water were considered for the measurement of size.^[6]

 

2.2.2.4 SEM:

The prepared samples of cubosomes are coated with a gold film under vaccum for 2min. The specimens are transferred to an ISI ABT SX-40A scanning electron microscope and digital images captured.

 

2.2.2.5 Determination of Percentage Drug Content:

1ml of dispersion was pipetted from the dispersion and was further diluted with pH 7.4 phosphate buffer saline and the samples were analyzed spectrophotometrically at 259nm.^[6]

 

2.2.2.6 Determination of Percentage Entrapment Efficiency:

The %EE of the vesicles was determined using centrifugation technique. The vesicular dispersion was centrifuged for 20 min. Supernatant containing unentrapped drug was withdrawn and measured UV spectrophotometricaly at 259nm against phosphate buffer saline pH 7.4. The amount of drug entrapped in liposomes was determined by:^[7]

                    T-C

% EE = ------------- X 100

                        C

Where,

T = Total amount of drug calculated in both supernatant and sediment.

C = Drug in supernatant.

 

2.2.2.7 In Vitro Drug Release:

In vitro drug release was measured using Franz diffusion cell. 50mg ketoprofen containing cubosomal dispersion was placed on one side of egg membrane in a vertical franz diffusion cell. Other side of membrane was in contact with the dissolution medium phosphate buffer saline of pH 7.4. Entire dissolution assembly was placed on a magnetic stirrer at temperature of 37°C. Aliquots of dissolution medium was withdrawn at different time intervals for 8hr. Drug concentration in the dissolution medium were determined by UV spectrophotometry at 259nm.^[8]

 

2.2.3 Preparation 0f Cubosomal Gel:

The cubosomal gel was obtained by addition of weighted amount of carbomer (1% w/w) in distilled water and kept for half day for getting swelling of carbomer and then add triethanolamine drop by drop up to pH 7. Propylene glycol is added to adjust the consistency. The obtained gel was then diluted with an appropriate amount of cubosomes dispersion in the ratio between the dispersion and the gel was 2:1 w/w.^[9]

 

2.2.4 Evaluation of Cubosomal Gel:

2.2.4.1 Appearance:

About 1 week after preparation, the dispersions were visually assessed for optical appearance (e.g., colour, turbidity, homogeneity, presence of macroscopic particles).^[9]

 

2.2.4.2 P^H

P^H of all formulations is determined by using digital pH meter by immersing the electrode in gel formulation and pH was measured.^[10]

 

2.2.4.3 Drug content:

1g of the prepared gel was mixed with 100ml of methanol. Aliquots of different concentration were prepared by suitable dilutions after filtering the stock solution and analyzed using UV.^[11]

 

2.2.4.4 Ex- vivo Skin Permeation Study:

In vitro skin permeation studies were performed using goat ear skin. The superficial skin was collected from the back of goat ear and the hair on the skin was removed. Skin was then mounted in a modified Franz diffusion cell, which is kept at 37ºC. Weighed quantity of cubosomal gel was then spreaded on the stratum corneum side of skin (donor compartment) and dermis side was facing receptor compartment. Receptor compartment contains 25ml of pH 7.4 phosphate buffer and after every one hour 1ml of sample was taken and replaced with the same volume of phosphate buffer. After 6 hours sampling, absorbance was measured at 259 nm against blank of pH 7.4 phosphate buffer by UV spectrophotometer. And the percentage drug permeated was calculated.^[12]

 

2.2.4.5 In-vitro Anti-inflammatory Activity:

The anti-inflammatory activity of cubosomal gel was studied by using inhibition of albumin denaturation technique. The reaction mixture (5ml) consisted of 4.5 ml of bovine serum albumin (5% aqueous solution) and 0.5ml of cubosomal gel, pH was adjusted at 6.3 using a small amount of 1N Hydrochloric acid. The samples were incubated at 37°C for 20 min and then heated at 57°C for 3 min. After cooling the sample, 2.5ml of phosphate buffer solution was added into each test tube. Turbidity was measured spectrophotometrically at 259 nm. For control tests; 0.5ml of distilled water was used instead of cubosomal gel. The percentage inhibition of protein denaturation was calculated as follows;^[13]

 

Percentage inhibition = (Abs _Control–Abs _Sample) X 100/ Abs _control

 

2.2.4.6 In-vitro Skin Irritation Test:

HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane) Test:

Incubated eggs of 9 days were collected from hatchery, shells were removed carefully using forceps. Test sample is applied directly to the CAM. Allow the sample for exposure to the CAM for atleast 300 second. The end point is measured by the visual inspections.^[14]

 

2.2.4.7 Release Kinetics:

Kinetic study was carried out by fitting the in vitro drug release data into Zero order, First order, Higuchi model, Hixon-Crowell Cube Root Law model and Korsmeyer- Peppas models. The best outfit model was confirmed by the value of R² which is near to 1.^[15]

 

2.2.4.8 Stability Studies:

Accelerated stability studies for optimized gel formulation (D2) were conducted as per ICH guidelines at 40°C ± 2°C/75% ± 5% RH at sampling intervals of 0, 30, 60 and 90 days respectively. The drug content pH and drug release are determined periodically.^[16]

 

3.   RESULT AND DISCUSSION:

3.1 Analytical method:

a) Determination of λ max:

The 10µg/ml sample was prepared and scanned between 200 to 400nm. The drug showed maximum absorption at 259nm. So, the λ max of Ketoprofen was found to be 259nm.

 

b) Calibration Curve:

Standard calibration curve data was given in table no.2. From fig. No. 1 y intercept and R2 value was found to be 0.021 and 0.995 respectively.

 

Table 2: Standard calibration curve data of Ketoprofen

 

Fig. 1: Calibration curve of ketoprofen

 

3.2 FTIR:

There were no significant changes in the frequency of the functional groups of Ketoprofen. So, the drug was compatible with Poloxamer, GMO and Carbopol.

 

 

Fig. 2: FTIR Spectra of Ketoprofen (sample)

 

3.3 DSC studies:

The melting point remains almost the same, indicated that the drug and excipients are compatible with each other.

 

3.4 Evaluation of Ketoprofen Cubosomes:

3.4.1 Optical Microscopy:

Images obtained under an optical microscope confirmed the formation of the crystal structures. Showed in figure no.3.It was found that the formed crystals were spherical and some are in rod shape.

 

Fig. 3: Microscopic view of Cubosome

 

3.4.2 Particle Size and Polydispersity Index:

The mean particle size and particle size distributions were determined by Malvern nano Zeta sizer instrument. The result is given in the table no.3.

 

Table 3: Particle Size and Polydispersity Index of Cubosome

Formulation	Average Particle size (nm)	Polydispersity index (PDI)
F1	488 ± 3.2	3.16 ± 0.1
F2	631 ± 2.4	4.05 ± 0.0
F3	679 ± 2.0	1.00 ± 0.0
F4	740 ± 1.9	1.81 ± 0.1
F5	650 ± 2.2	0.59 ± 0.1
F6	647 ± 3.7	0.91 ± 0.0
F7	453 ± 1.5	0.71 ± 0.0
F8	409 ± 3.0	1.24 ± 0.1
F9	655 ± 2.1	2.03 ± 0.0

 

3.4.3 Drug Content Estimation of Cubosome:

The % drug content in various formulations ranged from 90.68-95.38%. The drug content data revealed that there was no significant difference in the uniformity of the drug content in the formulations. So, it indicated that Ketoprofen was uniformly distributed in vesicular dispersions.

 

3.4.4 Drug Entrapment Studies of Cubosome:

Drug entrapment efficiency was determined in order to make sure that the added amount of Ketoprofen is present in the cubosome dispersion. The EE of all batches is in the range of 74.93 ± 0.903 - 92.10 ± 0.250. The highest EE was found in the batch F₇, consisted of 2.5 g of GMO, and 0.3g of poloxamer 407. The EE of Ketoprofen into cubic nanoparticles was dependent on the concentration of GMO. The result showed that the EE increased, as the amount of lipid and surfactant increased. Increasing amount of GMO was bound to increase the % of EE because of the increased concentration of mono-, di-, and triglycerides, which act as solubilizing agents for ketoprofen and provide more space to accommodate excessive drugs. This effect may be observed due to the increased viscosity of the medium, because increasing the amount of lipid resulted in faster solidification of the cubosomal nanoparticles, which would prevent drug diffusion to the external phase of the medium. As the percentage of emulsifier increased, part of the ketoprofen was incorporated in the surfactant layer at the surface of the cubosomes, leading to a high entrapment efficacy.

 

3.4.5 In Vitro Drug Release Studies of Cubosome:

The In vitro release characteristics of cubosomal dispersions shows that the drug release is directly proportional to the concentration of GMO and inversely proportional to the concentration of P-407 i.e. the cubosomes showed decrease in percent drug release when using of lower concentration of GMO and higher concentration of P-407 polymer. Here F7 has higher GMO concentration and lower Poloxamer 407 concentration, and it also showed higher percentage of drug release ie, 86.87% .

 

3.5 Evaluation of Cubosomal Gel:

From the drug content, drug entrapment and drug release study, its found that F7 is the best formulation. So it was selected and formulated to gel. Figure no.4.

 

Fig. 4: SEM image of Cubosome

 

3.5.1 Appearance:

It was determined by visual inspection. All the formulations were found to be homogenous.

 

3.5.2 p^H

The pH was found to be 5.7, which was close to skin pH.

 

 

3.5.3 Drug Content Estimation of Cubosomal Gel:

Drug content of the gel formulations was found to be 95±0.583%. (The reading is an average of 3 determinations).

 

3.5.4 Ex- vivo Skin Permeation Study:

The gel prepared using optimized cubosomal dispersion [F7] was used for ex-vivo permeation study using goat’s ear skin and showed 87.2% permeation through the skin. It is shown in table no:5.

 

Table 4: ex-vivo permeation study of cubosomal gel of optimized formulation

 

3.5.5 In Vitro Anti-inflammatory activity:

The cubosomal gel was analyzed for its anti inflammatory activity. Denaturation of proteins is a well documented cause of inflammation. From the results of present study it can be stated that the ketoprofen cubosomal gel is effective in inhibiting heat induced albumin denaturation. The percentage inhibition was found to be 70.77 %.

 

3.5.6 In-Vitro Skin Irritation Test:

The in-vitro skin irritation test was performed using HET-CAM. The Cubosomal gel formulation was found to be free of irritation and is safe. The observations shows absence of Haemorrhage, Lysis and Coagulation.

 

3.5.7 Kinetic Study of Ketoprofen Cubosomal Gel:

The kinetic study of the optimized formulation (F7) was also carried out and found that the formulation undergo zero order kinetics. The mechanism of drug release was found to be Higuchi model. The results are shown in table 6 and figure 9.

 

Table 5: Kinetic profile of Cubosomal gel

 

 

 

 

 

3.5.8 STABILITY STUDY:

P^H, Drug content and drug release values are analyzed periodically as per ICH guidelines through accelerated stability studies for optimized gel formulation was shown in Table 7.

 

Table 6: Stability study of Cubosomal gel

Time in days	pH	Drug content	Drug release
0	5.7	95	87.20
30	5.5	94	85.38
60	5.4	92	82.56
90	5.3	90	81.27

 

4.    SUMMARY:

Cubosomes can be prepared by simple combination of biologically compatible lipids (GMO) and water and are thus more suited for pharmaceutical and body tissue. Identification of drug was studied by IR spectral analysis. The Ketoprofen spectrum was compared with standard reference spectrum. There was no significant change in functional groups. The finger print region has not changed significantly. So the drug was identified as Ketoprofen.

 

The organoleptic evaluations of Ketoprofen were done. Its colour was found to be white or almost white. It was found to be odourless or almost odourless. Its appearance was found to be crystalline powder. The melting point determination of drug was also studied and it was found to be 97ºC by capillary tube method. The solubility studies of drug were also studied. The drug was found to be very slightly soluble in water and soluble in phosphate buffer pH 7.4, methanol, and chloroform. The analytical methods like absorption maximum, calibration curve of drug and compatibility studies were studied. The absorbance maximum of Ketoprofen in Phosphate Buffer pH 7.4 was measured as 259 nm. The cubosomal gel was analyzed for its anti inflammatory activity. Denaturation of proteins is a well documented cause of inflammation. From the results of present study it can be stated that the ketoprofen cubosomal gel is effective in inhibiting heat induced albumin denaturation. The percentage inhibition was found to be 70.77%. The in-vitro skin irritation test was performed using HET-CAM. The mechanism of drug release was found to be Higuchi model . The stability studies were carried out as per ICH guidelines. Thus it can be concluded that the formulation will provides controlled delivery of the drug in human via the noninvasive skin route with more sustaining, less frequent dosing and with more bioavailability when compared to oral delivery.

 

5.    ACKNOWLEDGEMENT:

I hereby acknowledge the support given by Nazareth College of Pharmacy, Othera, Thiruvalla.

 

6.    REFERENCES:

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15.  Rakesh P. Patel. Formulation and Evaluation of Carbopol Gel Containing Liposomes of Ketoconazole. International Journal Of Applied Pharmaceutics. 2011; 3(3): 1-9.

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Received on 04.02.2020            Modified on 21.03.2020

Accepted on 09.05.2020         © RJPT All right reserved