Novel Drug Delivery System (NDDS) fulfils desirable characteristics that it should deliver drug at a rate directed by need of body, over period of treatment & should channel active entity at site of action. Conventional dosage forms including prolonged released dosage forms unable to fulfilled none of these desired characteristics.¹ Vesicles have become the vehicle of choice in drug delivery system called Vesicular Drug Delivery System.”, e.g. liposomes, Niosomes, Pharmacosomes etc.² Liposomes are small artificial vesicles of spherical shape that can be created from cholesterol and natural non-toxic phospholipids. Due to their size and hydrophobic and hydrophilic character(besides biocompatibility), liposomes are promising systems for drug delivery.³ “Liposome  is  simple  microscopic  vesicles  in  which  an  aqueous  volume  is  entirely  enclosed  by  a  membrane  composed  of  lipid  molecule.⁴ Etoposide (ETP) is an antineoplastic agent, which acts by forming a ternary complex with topoisomerase II and DNA, causing DNA breaks and cell death. It is part of the first line therapy for small cell lung carcinoma and drug-resistant testicular cancer. However, strong lipophilicity is a major limitation in the administration of ETP, and vehicles needed as solubilizes have often been associated with adverse effects.⁵

MATERIALS

Etoposide was obtained from Alkem Pvt Mumbai, Cholesterol and Phosphatidylcholine procured from SD fine chemicals Mumbai. Other chemicals and the reagents used were of analytical grade.

METHODOLOGY

Preparation of Liposomes

Liposomes were prepared by physical dispersion method using different ratio of lipids.     In this method the lipids were dissolved in chloroform. This solution of lipids in chloroform was spread over flat bottom conical flask. The solution was then evaporated at room temperature without disturbing the solution. The hydration of lipid film form was carried out with aqueous medium phosphate buffer (pH 7.4). For this the flask was inclined to one side and aqueous medium containing drug to be entrapped was introduced down the side of flask and  flask was slowly returned to upright orientation. The fluid was allowed to run gently over lipid layer and flask was allowed to stand for 2 h at 37⁰C for complete swelling. After swelling, vesicles are harvested by swirling the contents of flask to yield milky white suspension. Then formulations were subjected to centrifugation. Different batches of liposomes were prepared in order to select an optimum formula. All batches of liposomes were prepared as per the general method described above.⁶

Drug Entrapment Efficiency of Liposomes

Entrapment efficiency of liposomes was determined by centrifugation method. Aliquots (1 ml) of liposomal dispersion were subjected to centrifugation on a laboratory centrifuge (Remi R4C) at 3500 rpm for a period of 90 min. The clear supernatants were removed carefully to separate non-entrapped Etoposide and absorbance recorded at 245nm. The sediment in the centrifugation tube was diluted to 100 ml with phosphate buffer pH 7.4 and the absorbance of this solution was recorded at 245nm.⁷

Amount of Etoposide in supernatant and sediment gave a total amount of Etoposide in 1 ml dispersion.

% entrapment of drug was calculated by the following formula

Particle Size Analysis

All the prepared batches of liposomes were viewed under microscope to study their size. Size of liposomal vesicles from each batch was measured at different location on slide by taking a small drop of liposomal dispersion on it and average size of liposomal vesicles were determined. ⁸

SEM Analysis

The morphology of liposomes was studied by a scanning electron microscope. For this purpose, the sample was lyophilized and placed on aluminium stubs and the surface was coated with a layer of gold particles using a sputter coater. The shape of the liposomes was determined by scanning electron microscopy (SEM) (XL30, Philips, the Netherlands) at 15 kV and 750mA.⁹

Zeta Potential (ZP)

Zeta potential (ZP) is a physical property that controls electrostatic interactions in particle dispersions and is essential in understanding the stability of colloidal dispersions. It is identified as the difference in potential between the particle and its ionic atmosphere surrounding the medium and is measured in the plane of shear. A ZP value of ± 30 mV is generally chosen to deduce particle stability, with an absolute value greater than 30 mV designated a stable condition, whereas a low zeta potential value of less than 30 mV indicates a condition toward aggregation, instability, flocculation, or coagulation. There are many factors affecting the stability of wound-dressing materials, including pH, temperature, instability radiation, concentration, the ionic strength of the solution, and the nature of the surface ligands.¹⁰

In Vitro Drug Release Study

The release studies were carried out in 10 ml Franz diffusion cell containing 10 ml Phosphate buffer. Phosphate buffer pH 7.4 (10ml) was placed in a 10 ml beaker. The beaker was assembled on a magnetic stirrer and the medium was equilibrated at 37±5⁰C. Dialysis membrane was taken and one end of the membrane was sealed. After separation of non-entrapped Etoposide liposomal dispersion was filled in the dialysis membrane and other end was closed. The dialysis membrane containing the sample was suspended in the medium.  1sml of aliquots were withdrawn at specific intervals, filtered after withdrawal and the apparatus was immediately replenished with same quantity of fresh buffer medium.¹¹

Drug Release Kinetics 

The models used were zero order (equation 1) First order (equation 2) and Higuchi model (equation 3) and Korsmeyer Peppas model (equation 4).  

i) Zero Order Kinetics: 

R =     Ko t                                              -- (1)

    R=cumulative percent drug 

    Ko=zero order rate constant

ii) First Order Kinetics

log C = log Co –K ₁ t /2.303                 -- (2)

Where C = cumulative percent drug 

  K ₁ = first order rate constant

iii) Higuchi Model

R = K _H   t ^0.5                                            -- (3)

Where R = cumulative percent drug

K _{H  =}  higuchi model rate constant

iv) Korsmeyer Peppas Model:

M t / M ?   = K _k t ⁿ  

log M t / M ? = log K _{k  +} n log t        -- (4)

Where K _{k  =}  Korsmeyer Peppas rate constant

‘M t / M ?’   is the fractional drug, n = diffusional exponent, which characterizes the mechanism of drug. The obtained regression co-efficient (which neared 0.999) was used to understand the pattern of the drug from the Liposomes.¹²

Stability Studies:

The success of an effective formulation can be evaluated only through stability studies. The purpose of stability testing is to obtain a stable product which assures its safety and efficacy up to the end of shelf life at defined storage conditions and peak profile. The prepared Etoposide liposomes were placed on plastic tubes containing desiccant and stored at ambient conditions, such as at room temperature, 40±2^oc and refrigerator 2-8^oc for a period of 90 days.¹³

RESULTS AND DISCUSSION

Drug-Excipient Compatibility Studies (FT-IR)

The compatibility between the drug and the selected lipid and other excipients was evaluated using FTIR peak matching method. There was no appearance or disappearance of peaks in the drug-lipid mixture, which confirmed the absence of any chemical interaction between the drug, lipid and other chemicals.

       
           
       
Fig-2: FTIR Analysis of Optimized Formulation

Vesicle Shape: Vesicle shape of the prepared formulation was found to be spherical from the SEM (scanning electron microscope) analysis at 15.00kV.

Vesicle Size:

       
           
       
Fig-3: Particle Size of Etoposide Liposomes

SEM Analysis

All the three batches of formulation F6 were found to release the drug in 8 h.  The cumulative percentage release was found to be 96.58 %.

Kinetic Modelling of Drug Release

All the Eight formulation of prepared matrix tablets of Etoposide were subjected to in-vitro release studies these studies were carried out using dissolution apparatus.

The results obtaining in vitro release studies were plotted in different model of data treatment as follows:

1.   Cumulative percent drug released vs. time (zero order rate kinetics)

2.   Log cumulative percent drug retained vs. time (First Order rate Kinetics)

3. Cumulative percent drug released vs. square root of time (Higuchi’s 

     Classical Diffusion Equation)

4.   Log of cumulative % release Vs log time (Peppas Exponential Equation)

The table indicates that r² values are higher for Higuchi’s model compared for all the liposomes. Hence Etoposide release from all the liposomes followed diffusion rate controlled mechanism.

Stability Studies

There was no significant change in physical and chemical properties of the liposomes of formulation F-6 after 3 months. Parameters quantified at various time intervals were shown