Formulation and Evaluation of Dexamethasone Loaded Cubosomes

 

Thoutreddy Rajani^1,2*, Gullapudi Mahesh¹, Bonepally Chandra Shekhar Reddy¹

¹Department of Pharmaceutics, Vaagdevi College of Pharmacy, Kakatiya University, Kishanpura,

Hanamkonda-506001, Telangana, India.

²Research Scholar, Koneru Laxmaiah Education Foundation, Guntur, Andhra Pradesh, 522502

 

ABSTRACT:

Cubosomes are altered cubic phase systems, which are emerging as promising drug delivery system for the delivery of both hydrophilic and lipophilc drugs. Dexamethasone is a lipophilic steroidal drug with poor hydrophilicity. Lipophilic drugs like Dexamethasone can be successfully administered by use of novel transdermal systems like cubosomes, nanoparticles, liposomes, implants etc. Controlled drug delivery, increased time scale of action, preventing the necessity of frequent parenteral and ophthalmic admisitration is enhanced by loading Dexamethasone in the form of cubosomes. The main aim of present research was to encapsulate Dexamethasone in cubosomes for sustained drug release. Dexamethasone loaded cubosomes were prepared by top-down technique using Glyceryl Mono Oleate and Poloxamer 407 in different ratios. The prepared formulations were subjected to evaluation studies for excipient compatability,  particle size, zeta potential, drug content, entrapment efficiency and In vitro drug release. The maximum entrapment efficiency was found as 96% with vesicle size as 119.4 nm, charge as -22.1±5.66 mV, Poly Dispersity Index as 0.153 and In vitro drug release as 92.12% by dialysis bag method over 24hrs. Stability studies were also conducted for the formulations as per protocol mentioned in ICH guidelines. These results suggest that the cubosomal formulation F6 is suitable for the delivery of Dexamethasone.

 

 

1. INTRODUCTION:

Dexamethasone (C₂₂H₂₉FO₅) is a strong synthetic glucocorticoid steroidal drug used to treat various inflammatory and autoimmune conditions like Rheumatoid arthritis, edema, nasal and opthalmic allergies.  It is poorly water soluble and is lipophilic in nature. Parenteral and Ophthalmic routes are commonly used to administer Dexamethasone. It has half-life of about 30-52 hours and 70% of protein binding¹.

 

Oral usage of glucocorticoids causes numerous adverse and toxic effects like stomach upset, disturbances in electrolytic balance, muscle atrophy, negative protein balance (catabolism), enhanced appetite causing significant weight gain etc². Use of transdermal routes eliminates the above side effects, increases patient compliance and maintains the plasma drug level for a longer period of time³.

 

Lipophilic drugs like Dexamethasone can be successfully administered by use of novel transdermal systems like cubosomes, gels, nanoparticles, liposomes, implants etc. Controlled drug delivery, increased time scale of action, preventing the necessity of frequent parenteral and ophthalmic admisitration is enhanced by loading Dexamethasone in the form of cubosomes¹.

 

Cubosomes are discrete, sub-micron, nanostructured particles of cubic liquid crystalline phase. These are microstructure particles containing surfactants with proper ratio of water. They are particles with a solid-like rheology that provides unique properties of practical interest⁴. Cubosomes size ranges from 10-500nm in diameter. They appear like dots, square shaped, slightly spherical, where each dot corresponds to the presence of pore size of 5-10nm. Cubosomes are typically produced by high-energy dispersion of bulk cubic phase, followed by colloidal stabilization using polymeric surfactants. After formation of the cubosomes, the dispersion is formulated into a suitable dosage form⁵.

 

Cubosomes have great potential in formulating nano sized particulate systems for topical delivery owing to their best advantages such as high drug payload due to high internal surface area, low viscosity and can exist at almost any dilution level. Structurally these are lipid vesicles formed from amphiphilic building blocks, which mimics bio membranes that can be used for carrier potential of hydrophilic, lipophilic and amphiphilic drugs as compared to free drug directly delivered to the particular site of action, thus allowing drug targeting and the sustained or controlled release of conventional medicines⁶. However the efficiency of cubosomes alone to deliver the drugs in a controlled way is less when compared with cubosomes incorporated with polymers⁷. The high viscosity of these cubosomes is a challenging task during large scale preparation in industries⁸.

 

Cubosomes can be administrated in many ways such as oral, percutaneous, intravenous route. Cubic phase is unique and desirable because of its mesoscale structure: a twisted lipid bilayer, separating two continuous but non-intersecting water regions. The tortuous structure of bulk cubic phase provides controlled release of solubilized active ingredients, while cubosomes exhibit burst release because of their sub-micron length scales⁹.

 

With the aim of formulating a sustained delivery, cubosomes of Tretinoin, a lipophilic drug were prepared using bottom up technique and were incorporated in the form of a gel. The results depicted the efficacy of cubosmes to deliver the drug with good retention property on the skin in treatment of acne¹⁰.  Localized eefctive penetration of antiinflaammatory agent, Etodolac was successfully developed by formulating it in the from of cubosomal gel. Salah et al evaluated the efficiency of etodolac to deliver the drug in treatment of arthritis¹¹. The purpose of present study was to develop Dexamethsone loaded Cubosomes to sustain the drug release, increase penetration efficiency, stability and to reduce side effects caused by conventional routes of administration of the drug.

 

2. MATERIALS AND METHODS:

Dexamethasone was received as a gift sample from Yarrow Chem. Products, Mumbai, India. Poloxamer 407 was obtained from Daewoong Pharmaceuticals Ltd, Hyderabad. Glyceryl Mono Oleate was procured as a gift sample from Mohini Organics Pvt. Limited, Mumbai.

 

2.1 Preparation of Dexamethasone loaded Cubosomes:

The method used for the preparation of cubosomes was top-down method⁹. Varying concentrations of Glceryl Mono Oleate (GMO) along with Poloxamer 407 as shown in Table 1, were accurately weighed and heated on an electric water bath at a temperature of 40 to 45ºC until Poloxamer 407 completely dissolved in GMO. To the above solution drug was added and was mixed well. The clear lipid solution obtained was added slowly to distilled water and was subjected to probe sonication for 10 min. The resultant solution obtained was white opaque dispersion without presence of any aggregates. The prepared dispersions were stored in closed glass vials at room temperature for 72hrs, protected from light and later evaluation was carried out.

 

2.2 Evaluation of Dexamethasone Cubosomes:

2.2.1 Drug-excipient compatability study:

Drug-excipient interaction was studied before developing the formulation by using FTIR spectroscopy, which is one of the most important analyses to investigate the stability of formulation, and molecular interactions between the drug and the excipients used¹². Fourier-transform infrared spectroscopy (FT-IR) measurements were performed using FTIR spectrophotometer using KBr disc method. The samples were scanned over the range of 4000 to 400^cm-1.

 

Table 1: Formulation of Cubosomes using GMO and Poloxamer 407.

 

2.2.2 Surface morphology:

The morphology of cubosomes was determined using Transmission Electron Microscopy (TEM)¹³. TEM gives a morphological image of the globules. The samples were examined at suitable accelerating voltage of 20 kV, at different magnification.

 

2.2.3 Particle size analysis and zeta potential:

The particle size and zeta potential of cubosomes were determined by dynamic light scattering technique using Malvern particle size analyzer. Samples were diluted in particle-free purified water and measured at 25ºC. Zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in dispersion system.  The zeta potential is a key indicator of the stability of dispersions.

 

2.2.4 Entrapment efficiency:

For the determination of entrapment efficiency, the cubosomal dispersions were subjected to centrifugation at 15000 rpm for 30 min. The resulting solution was then separated and supernatant liquid was collected. The collected supernatant was then diluted appropriately and estimated using UV visible spectrophotometer at 241 nm.

 

The percentage of encapsulation efficiency (%EE) was determined by the following equation:

 

                   Total drug- Free drug

%  EE =   –––––––––––––––––––  ×  100

                          Total drug

 

2.2.5 In Vitro drug release studies:

In vitro drug release studies were carried out using dialysis bag which was placed on a magnetic stirrer and temperature was adjusted to 37±0.5ºC. One end of the cylinder was covered with gelatin membrane, which was previously soaked in warm water. The diffusion cell was placed in a 50mL Borosil beaker that served as the receptor cell. The temperature in the diffusion and receptor cells was maintained at 37ºC, with the help of a thermostat. Phosphate buffer pH 7.4 was placed in the receptor cell. Cubosomal formulation was placed on the dialysis membrane, which was in contact with receptor medium. Samples were withdrawn from the receptor cell at specified time intervals of 1, 2, 3, 4, 5, 6, 12 and 24 hrs. Each time immediately after the removal of the sample, the medium was compensated with fresh phosphate buffer (pH 7.4). The samples were analyzed for drug content using a UV spectrophotometer at 241 nm.

2.2.6 Release Kinetics:

The optimized cubosomal formulation (F6) was studied for release kinetics. Coefficient of correlation values were calculated for the linear curves obtained by regression analysis of the plots.

 

2.2.7 Stability studies:

Stability investigations of cubosomal formulation have been performed based on the ICH guidelines at 40°C ± 2°C/75% ± 5% RH and at 25°C ± 2°C/60% ± 5% RH¹⁴. 5 mL of cubosomal suspension was filled in vials and was kept in stability chambers (Thermo Scientific Equipments, India) for 25ºC, 40ºC and 4± 2ºC in refrigerator for 4 weeks. The samples of cubosomal formulations were withdrawn at regular time periods of 0, 1, 2, 3 and 4 weeks and examined for precipitation, colour changes, drug content, entrapment efficiency, particle size, phase separation and drug release. The appearance and phase separation which indicate the stability of the cubosomal suspension were determined at regular time points.

 

3 RESULTS AND DISCUSSION:

3.1 Solubility studies:

Dexamethasone has shown highest solubility in methanol compared to phosphate buffer  and water.  The solubility of Dexamethasone in methanol is 28.4 µg/mL where as in case of water the solubility is 6.8 µg/mL. It is soluble in anhydrous ethanol and slightly soluble in Dichloromethane.  

 

3.2 Drug identification by FTIR:

The interaction study between the drug and the excipients as well as optimized formulation was evaluated using FTIR spectrophotometer as shown in Table 2. Dexamethasone alone showed the principal peaks and were observed at wavenumbers at 2931.93 cm⁻1 due to aliphatic C-H stretching, O-H stretching at 3393.58 cm⁻1, C=O stretching at 1712.36 cm−¹, C-N stretching at 1665.58 cm^-1, C=C aromatic stretching at 1431.82 cm^-1, C-O-C stretching at 1136.10 cm^-1 and C-N stretching at 1081.66 cm^-1. Similar peaks were observed in spectra of combination of excipients and in optimized formulation along with absence of interfering peaks indicating that there is no unwanted reaction between Dexamethasone and other excipients used in the study.   

 

 

 

Table 2: FTIR peak values of Dexamethasone and optimized cubosomal formulation.

 

 

 

3.3 Characterization of Cubosomes:

3.3.1 Surface Morphology of Cubosomes:

The surface morphology of the cubosomes was determined using Transmission electron microscopy (TEM). It was observed that the obtained cubosomes have a smooth surface and cubic in shape (Fig 1).

 

 

Fig 1: TEM  image of Cubosomes

 

 

Result quality                                                     Good                                  

Fig 2: Particle size and PDI values of Dexamethasone loaded cubosomes.

 

 

 

Fig 3: Zeta potential value of Dexamethasone loaded cubosomes.

3.3.2 Particle Size and Zeta Potential of Cubosomes:

The particle size and zeta potential of cubosomes were determined by dynamic light scattering technique using Malvern particle size analyzer. From Fig 2, it was found that the diameter of cubosomes was found to be in the range of 10 to 500nm and the average particle size was found to be 119.4nm. From the Fig 3, the zeta potential was found to be -22.1mV, which indicates that cubosomes were stable.

 

3.3.3 Entrapment Efficiency:

The entrapment efficiency of the cubosomal formulations was found to be in the range of 80% to 96%. Entrapment efficiency of the cubosomes was found to increase by increasing GMO concentration of 8% to 20%. Hence formulation F6 was considered as optimized formulation based on high entrapment and stability.

 

3.3.4 Drug release from cubosomes by Dialysis bag method:

In vitro release study of Dexamethasone from cubosomes was carried out by dialysis bag method and the following results were observed (Table 3). This study showed that all the formulations exhibited good release of drug from the cubosomal vesicles but in different release behaviour. Only the formulations F5 and F6 had shown maximum drug release in sustained release behaviour with different rate of release. The cubosomes prepared with the addition of 16 %w/v Monolein (F5) and 18 % w/v (F6) showed sustained drug release than other formulations, i.e., 88.20% and 92.12% drug release in 24hrs. So these two formulations can be incorporated into the gel base for better sustained, site specific and local action.

 

In vitro diffusion studies were carried out with the prepared cubosomes as shown in Table  4. The release data indicates that all formulations sustained drug release for about 4hrs.       

 

3.3.5 Release Kinetics:

The optimized cubosomal formulation was studied for release kinetics as shown in Table 4. It was found that F6 formulation showed first order drug release.

 

Table 3: Cumulative percent drug release of Dexamethasone loaded cubosomal formulations.

#All the values are expressed as mean ±SD, n=3

 

 

 

Table 4 : Release kinetics of optimized  formulation(F6).

Formulation code	R2 Value
	Zero order	First order	Higuchi Order	Korsemeyer-Peppas	n-value
F6	0.8641	0.9900	0.9284	0.9815	0.6331

 

Table 5 : Stability studies of Dexamethasone cubosomal suspension (F6).

 

 

 

3.3.6 Stability studies:

The stability studies were conducted for optimized formulation and physical properties were analysed periodically as per ICH guidelines as shown in Table 5.

 

4. CONCLUSION:

A satisfactory attempt was made to develop Dexamethasone cubosomes using GMO and Poloxamer 407 as polymer and were evaluated for compatibility and morphological studies. In vitro drug release study revealed that cubosomal formulation F6 containing 1% Poloxamer 407 and GMO concentration of 18% showed better release (92.12%) good entrapment efficiency (96%), good stability (-22.1mV) and showed mono-dispersity. It can be concluded that cubosomes are promising vehicle for delivery of Dexamethasone.

 

5. ACKNOWLEDGEMENTS:

We are thankful to Yarrow Chem. Products, Mumbai for providing gift sample of Dexamethasone, Mohini Organics Pvt. Limited, Mumbai for providing gift sample of Glyceryl monooleate and Daewoong Pharmaceuticals, Hyderabad for providing Poloxamer 407 for this work. We also thank Vaagdevi College of Pharmacy for the support in completion of the above work.

 

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Accepted on 11.09.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):709-714.