Formulation development of Temozolomide liposomal formulation in the treatment of Glioma

 

Swapna Velivela*, Nikunja B Pati, B. Ravindra Babu

Pulla Reddy Institute of Pharmacy, Domadugu (V), Gummadidala (M), Sangareddy Dist, Telangana 502313.

*Corresponding Author E-mail: swapna.velivela@gmail.com

 

ABSTRACT:

Temozolomide is an anti-cancer drug; it was encapsulated in liposomal intravenous application. To avoid the side effects and to target the drug to the specific site, we have formulated liposomal formulation of Temozolomide. The liposomal were prepared by dried thin film hydration technique using rotary evaporator with drug and Soya phosphatidyl choline as carrier. The prepared liposomes were characterized for size, shape, % entrapment efficiency, in-vitro drug release and physical stability. The evaluated batches showed good physicochemical characteristics. The maximum encapsulation efficiency of Temozolomide was achieved with formulation TMZ 6 with 40.19% and the in-vitro drug release is 64.94%. Based on the results it can be concluded that TMZ 6 was selected as optimized formulation and the optimized formulation Optimized formulation follows zero order release kinetics and follow super case II transport when it applied to Korsmeyer-Pepps model for mechanism of drug release.

 

KEYWORDS: Temozolomide, Anti-cancer drug, Film hydration technique, % Entrapment efficiency, In-vitro drug release.

 


INTRODUCTION:

Research has been focused in investigating a variety of drug delivery systems aimed at promoting better transport of active drugs by using colloidal drug delivery systems such as micro emulsions, solid lipid nano-particles and liposome’s for topical delivery1-4.  Liposomes are enclosed vesicles containing a lipid bi layer composed of unimers that usually have a hydrophilic head and a hydrophobic tail and are oriented so that the hydrophobic head groups are inside the bi layer5-6. Liposomes are highly biocompatible with low toxicity that helps in conniving drug delivery system with improved bioavailability6.

 

Hydrogels are 3-dimensional networks consisting of hydrophilic polymers that swell in aqueous solution retaining large amount of water without dissolving. Hydrogels formulated with cellulose have biodegradable properties, high permeation of active materials with high degree of swelling and no associated toxicity or irritation makes them as ideal polymers for delivery of drugs through transdermal route as delivery vehicles7-10. Temozolamide is a imidazotetrazine derivative acts by alkylate or methylate DNA which triggers the death of tumor cells. Temozolomide (TMZ), is a medication used to treat some brain tumors such as glioblastoma multiforme or anaplastic astrocytoma with elimination half-life of 1.8 hours11-12.

 

MATERIALS AND METHODS:

Temazolamide (TMZ) was a gift sample from Cipla, Hyderabad. Hydrogenated soya phosphotidylcholine, cholesterol and methoxy polyethylene glycol and other chemicals were purchased from Sigma Aldrich, Mumbai.

 

Preparation of Temazolamide liposomes by thin film hydration method13-15:

Six liposomal formulations were prepared by thin film hydration method using Temazolamide (TMZ) was a gift sample from Cipla, Hyderabad. Hydrogenated soya phosphatidylcholine, cholesterol and methoxy polyethylene glycol. Accurately weighed quantities of Hydrogenated soya phosphatidylcholine, cholesterol and mPEG-DSPE is a linear PEG phospholipid with saturated C18 stearoyl fatty acid (octadecanoic acid) and are dissolved in chloroform and methanol rotated in Rota evaporator by applying vacuum, until it forms a thin film. Required quantities of ammonium sulphate and glucose are dissolved in water for injection and it is added to the above thin film in round bottom flask and rotated until it forms milky white suspension. The above solution is homogenized for 15 cycles to reduce the particle size. The above solution is undergone 25 cycles of dialysis, by using sucrose (10%) to remove ammonia from the solution.  Required quantities of drug solution is added to the above solution in RBF (lipid solution) and rotated for 1 hour for the production of liposomes.

 

Table 1: Composition for different liposomal formulations

Formulation code

Lipid Molar Composition

(HSPC: mPEG-DSPE: Chol)

Temozolomide

Ammonium Sulphate

TMZ 1

50:0.5:25

15 mg/ml

30 mg/ml

TMZ 2

50:1:30

15 mg/ml

30 mg/ml

TMZ 3

50:2:35

15 mg/ml

30 mg/ml

TMZ 4

50:3:40

15 mg/ml

30 mg/ml

TMZ 5

50:4:45

15 mg/ml

30 mg/ml

TMZ 6

50:5:50

15 mg/ml

30 mg/ml

 

Characterization of liposomes:

All the liposomal formulations were evaluated for physicochemical properties, particle size analysis, polydispersity index, zeta potential analysis, SEM analysis, entrapment efficiency, assay, in-vitro drug release and stability studies.

 

Physico-chemical Characteristics14:

The liposomal formulations were evaluated for their physico-chemical properties and found to be colorless and odorless. The liposomal hydrogels were also found to be odorless, translucent and with neutral pH. The properties of formulations immediately and on 15 day did not show any differences in their properties indicating the physical stability of the formulations. The Physico-chemical properties of Temazolamide liposomal hydrogel complex suggest that it has a good potential for topical drug delivery.

 

Particle Size:

The mean particle size and size distribution were determined by photon correlation spectroscopy with a Zeta-sizer Nano ZS (Malvern, UK) at 25°C.

 

Scanning Electron Microscopy (SEM):

Scanning electron microscopy was used to characterize the surface morphology of the prepared vesicles. One drop of liposomal dispersion was mounted on a clear glass stub, air-dried, coated with Polaron E 5100 sputter coater (Polaron, Watford, United Kingdom), and visualized under a scanning electron microscope.

 

Entrapment Efficiency (EE)15:

Entrapped drug in the liposomes was estimated after removing the unentrapped drug. The unentrapped drug was separated by subjecting the dispersion to centrifugation in a cooling centrifuge at10000 rpm at a temperature of 4˚C for 10 minutes. The supernatant was removed and the pellet of liposomes was washed with 5 ml buffer to remove any unentrapped drug. The washing was combined with supernatant and was analyzed for drug content at 210 nm to calculate the entrapment efficiency.

 

In vitro drug release16-18:

In-vitro release of drug from liposomal formulation carried out by using dialysis membrane employing two side open ended cylinder. 4 ml of liposomal suspension containing known amount of drug was placed in a donor compartment. The two sides open cylinder was placed in 200 ml of phosphate buffer pH 7.4, maintained at 37OC and stirred with the help of a magnetic stirrer. 4 ml of release medium were withdrawn at different time intervals and the same volume was replaced with fresh phosphate buffer pH 7.4, to maintain sink conditions and these samples were analyzed by UV Spectrophotometry at a λ max of 210nm.Results were tabulated and graph was plotted as percentage drug release versus time for all six formulations.

 

Release Kinetics19-20:

The release data fitted in various release kinetic models like Zero order, first order, Higuchi and Korsemeyer-Peppas equations to know the mechanism of drug release kinetics.

 

RESULTS AND DISCUSSION:

Average size and size distribution:

The average size of the Temazolamide liposomal formulation was carried out using wet method. The average particle size of optimized formulation is 109nm.

 

Zeta potential:

Zeta potential report of liposomal solution which lies near to arbitrary value. The report shows good stability value for formulated liposomal solution.

 


Figure 1: Zeta Potential for Temozolomide Liposomal TMZ 6 formulation

 


Entrapment efficiency:

The amount of Temazolamide entrapped in the liposomes was determined by complete vesicle disruption followed by extraction with triton X-100. The percentage entrapment of Temazolamide was found to be maximum with TMZ6 formulation. The entrapment efficiency of drug increased when molar ratio of lipid cholesterol was increased. This may be due to increasing concentration of lipids which may increase lipophilic properties of lipid bilayer hence increase entrapment of lipophilic drug.

 

Table 2: Assay, % Entrapment efficiency and Zeta Potential values of prepared formulations

Formulation code

Assay

% Entrapment efficiency

Zeta potential (mV)

TMZ 1

97.2±0.52%

19.63%

-6

TMZ 2

98.1±0.23%

22.12%

-7

TMZ 3

98.5±0.13%

29.50%

-9

TMZ 4

98.4±1.22%

31.20%

-10

TMZ 5

98.5±0.88%

36.78%

-10

TMZ 6

99.2±0.64%

40.19%

-12

 

In vitro drug release:

The In vitro dissolution profiles of prepared formulations were determined by membrane diffusion method. The diffusion was carried out for a period of 48 hours in phosphate buffer pH 7.4. The maximum percent of drug release was found in TMZ6 formulation with 64.94% in 48 hours.

 

 

 


Figure 2: % Entrapment Efficiency of prepared liposomal formulations

 


Release kinetics:

The release kinetics of TMZ6 formulation was studied. Optimized formulation follows zero order release kinetics and follow super case II transport when it applied to Korsmeyer-Pepps model for mechanism of drug release.

 

Figure 3: Cumulative % Drug release of prepared liposomal formulations

 

CONCLUSION:

Temazolamide is an anti- cancer drug used in the treatment of glioma which consist many of the unwanted side effects.  To avoid these side effects, an attempt on temazolamide is done by preparing in the form of liposomal preparations with different compositions of lipid and cholesterol. Out of 6 formulations TMZ 6 formulation was selected as optimized formulation shows the significant drug is entrapped in liposomal vesicle to show desired effect. From the experimental results, it can be concluded that that the hydrogenated soya phosphatidyl, mPEG-DSPE and cholesterol were suitable carriers for the preparation of Temozolomide liposomes.

 

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Received on 03.02.2021            Modified on 14.03.2021           

Accepted on 07.04.2021      ©Asian Pharma Press All Right Reserved

Asian Journal of Pharmacy and Technology. 2021; 11(3):203-206.

DOI: 10.52711/2231-5713.2021.00033