Formulation Development, Characterization and In-vitro Evaluation of Tamoxifen Loaded Liposomes

Background: The study was aimed to prepare and evaluate tamoxifen loaded controlled release liposomes to reduce the side effects of tamoxifen during cancer treatment. Methods: Different tamoxifen loaded liposomes were prepared by modified ether injection (MEIM) and thin film hydration method (TFHM) under prescribed conditions. The prepared liposomes were characterized by using optical microscopy, evaluating encapsulation efficiency, in-vitro and ex-vivo diffusion studies by using dialysis membrane and chicken intestinal sac respectively. Results: The data revealed that all of the liposomes were spherical in shape and stable under three physical conditions i.e. 4, 25 and 37 ± 2°C temperatures and 60 ±5% relative humidity. Additionally most of the liposomes followed zero order and class II release kinetics. It was also observed that with the increase of phospholipids and cholesterol, entrapment efficiency of liposome vesicles increased thus giving a controlled release drug delivery system but further increase reduced this efficiency at a certain level. Conclusion: The formulated control release liposomes might be a good drug delivery system for target oriented drug delivery with minimum side effects of tamoxifen during cancer treatment. Original Research Article Hasan et al.; JPRI, 32(6): 64-82, 2020; Article no.JPRI.56540 65


INTRODUCTION
Tamoxifen is used very commonly for the treatment of estrogen receptor positive breast cancer which is considered a pioneering drug because of its ubiquitous application in both treatment and chemoprevention of breast cancer [1] and also reduction of the chances of breast cancer in high-risk patients [2]. It is generally administered through oral and parenteral route to treat patients but several potential problems arise uniquely because of the use of oral therapy and oncologists need to be aware about the drug interactions and bioavailability of tamoxifen [3]. Whitfield et al. has reported that tamoxifen has a relatively high biological half-life which is 5-7 days and 99% protein binding after administration which reduces the effectiveness of the drug. So there is a strong clinical need and market potential for a new drug delivery system [4].

Bozzuto and
Molinari [5] proposed that liposomal drug delivery system is a controlled targeted drug delivery system and has benefits like patient compliance, avoiding multiple dosing, increased bioavailability, fewer side effects these will help to overcome the problems associated with conventional system. Liposomes are considered the almost ideal drug-carrier system due to their similar morphology to cellular membranes and have ability to encapsulate both hydrophilic and lipophilic substances because of unique structural characteristics [6,7]. It is reported that a number of drugs have already been successfully encapsulated in liposomes, from antibacterials [8] and interferons to antitumor drugs such as doxorubicin [9] where it is proven that liposomes are useful in terms of biocompatibility, biodegradability, low toxicity, and can control bio distribution by changing the size, lipid composition, and physical characteristics [10,11,12].
In our study, modified ether injection method [13] and thin film hydration method [14] were used to prepare tamoxifen loaded liposomes where cholesterol was used to improve the liposomal delamination and to improve membrane fluidity, stability of the layers and reduce the permeability of water-soluble molecules through membranes. The presence of cholesterol in the lipid layers increased the stability and results in the formation of a hard membrane with similar liquid properties [15,16].
The purpose of this work was to formulate and evaluate a modern drug delivery system, tamoxifen loaded liposome to improve bioavailability, therapeutic effectiveness as well as reduce drug resistance, cytotoxicity and other unwanted effects.

Materials
Materials used for the preparation and characterization of tamoxifen loaded liposomes were listed in Table 1.

Preparation of Tamoxifen Loaded Liposomes by Modified Ether Injection Method (MEIM)
Cholesterol and phospholipids were dissolved in 8 mL diethyl ether and mixed with 2 mL methanol containing weighed quantity of tamoxifen. The resulting solution was injected using a micro syringe at a rate of 1 mL/min into 10 mL phosphate buffer (pH 7.4) hydrating solution at constant temperature (50-55ºC) with continuous stirring on magnetic stirrer at 300 RPM (MS 300 Hot plate Magnetic Stirrer, BANTE instruments, USA). As the lipid solution was injected slowly into the aqueous phase, the difference in temperature between phases caused rapid vaporization of ether, resulting in spontaneous vesicle formation of liposomes. All the formulations as per experimental design were prepared using similar procedure by addition of various quantities of phospholipids and cholesterol [17].

Preparation of Tamoxifen Loaded Liposomes by Thin Film Hydration Method (TFHM)
Cholesterol and phospholipids were dissolved in 8 mL chloroform and mixed with 2 mL methanol containing weighted quantity of tamoxifen and mixed together in a round bottom flask. Using the rotary flash evaporator, the organic solvents were removed at 45-50ºC at 120 RPM which leaves a thin layer of solid mixture on the wall of the flask. The dried film is then rehydrated with 20 mL of pH 7.4 phosphate buffer solution at the temperature of 60 ± 2ºC for a specified period of time (about 2-2.5 hours) with gentle agitation.

Formulation Design
For the preparation of tamoxifen loaded liposomes using central composite design of user defined factorial design was adopted to optimize the formulation parameters and to study the influence of independent formulation variables on dependent variables. Percent of drug entrapment efficiency and drug release were considered as dependent variables while phosphatidylcholine and cholesterol were taken as independent variables for liposomal formulations.
Nine experimental runs were designed (design Export® software-Trial Version 11 Stat-Ease Inc., MN) by selecting two parameters phosphatidylcholine and cholesterol amount at three levels each (low, medium and high) as independent variables where the amount of drug (10 mg) was kept constant for each batch shown in Table 2.

Preparation of Standard Curve
Standard curve of tamoxifen was prepared by dissolving 10 mg pure drug (hydrated with 3-4 drops of ethanol) up to 100 mL with phosphate buffer (pH 7.4 & 6.8) and acidic buffer (0.1N & 0.02N HCl) respectively. Afterwards these standard primary solutions were diluted with respective media to obtain varying concentration of stock solutions and absorbance was obtained at 237 nm by using UV spectrophotometer (UV mini-1240, Shimadzu Corporation, Japan).

Characterization of Physical Parameters of Liposomes
Prepared liposomal batches were monitored for their morphological attributes using optical microscope (at suitable magnification). Size distribution profile of liposomes was determined by light scattering based on laser diffraction method [21].

Ex-vivo Permeability Study by Using Chicken Intestinal Sac
Ex-vivo permeability studies were carried out through dissolution study conducted in a dissolution machine using USP II paddle apparatus by using chicken intestinal sacs. Total nine cleaned intestinal segments (each six cm length) were ringed with a phosphate buffer solution (pH 7.4) and 2 mL of liposomes suspension was placed in the sac which was then sealed at both ends. The sacs were dipped into the receptor compartment containing 1000 mL of phosphate buffer (pH 7.4) at 100 RPM and temperature was maintained at 37ºC [22]. 10 mL of the sample was withdrawn at predetermine intervals from each basket, filtered and vessel was replenished with fresh medium. Absorbance was taken by using spectrophotometer at 237 nm and the permeability study was checked for eight hours.

In-vitro Permeability Study by Using Cellulose Dialysis Membrane
In vitro permeability study was done using cellulose dialysis membrane (Spectrapor, USA) in USP II paddle method. Dialysis membrane was cut into nine cm in length and soaked in 500 mL distilled water at room temperature for 30 minutes to 1 hour to remove the sodium azide preserving agent. Then the membrane was rinsed thoroughly in distilled water and 2 mL of liposomes was placed in the membrane which was then sealed at both ends. The membrane was dipped into the receptor compartment containing 1000 mL of phosphate buffer (pH 7.4) dissolution medium. The dissolution was carried out based on the specification followed in ex vivo permeability study [23].

Interpretation of dissolution profile
Percent release of drug was obtained from the formulation of liposomes by comparing the absorbance value with the standard curve of tamoxifen [24].

Release kinetics
Data obtained from in vivorelease studies were fitted to various kinetic equations to find out the mechanism of drug release from the liposomes.

Zero-order kinetic model
Zero order described the system where the release rate of drug was independent of its concentration where data obtained from the invitro drug release studies were plotted as cumulative amount of drug released versus time [25].
This can be represented by the equation: Here, Q t = Release of drug at time't' Q 0 =Initial amount of drug in the solution at t=0 k 0 = First order release constant

First order kinetic model
This model was used to describe the absorption and elimination of drugs from liposomes where obtained data were placed as log cumulative percentage drug remaining verses time, which yield a straight line with slope = (K / 2.303) [26].
The drug release which followed the first order kinetic can be expressed by the equation: Here, C= Concentration of drug C 0 = Initial concentration of drug k= First order constant

Higuchi kinetic model
This model was used to describe drug release from matrix system where data obtained were plotted as cumulative percentage of drug release versus square root of time [27].
A form of Higuchi square root law was given by equation:

RESULTS AND DISCUSSION
The liposomal formulation containing tamoxifen were prepared by modified ether injection method (MEIM) and thin film hydration method (TFHM) using phosphatidylcholine and cholesterol with the purpose of evaluating the effect of phospholipids and cholesterol and their different level on physical and structural properties of liposomes.

Standard Curve of Tamoxifen
Standard curve of tamoxifen was prepared in acidic buffer (0.1N and 0.02N HCl) and phosphate buffer (pH 6.8 and pH 7.4) respectively. Absorbance of tamoxifen for different media was cited in Table 3. It was observed that R

Percent Drug Entrapment Efficiency (%DEE) of Liposomes
Total nine liposomal formulations of tamoxifen were prepared by following MEIM where percent of drug entrapment efficiencies of different formulations were in range of 60.12% to 86.83% and TFHM in range of 59.19% to 86.03% respectively. It was seen that formulation TEI-1 and TTFH-1 showed lowest tamoxifen entrapment efficiency due to the lowest level of phosphatidylcholine and cholesterol in the formulation. On the other hand, TEI-9 and TTFH-9 revealed highest tamoxifen entrapment efficiency because of highest amount of phosphatidylcholine and cholesterol present in the formulation for both methods [30].
Additionally, having very low phospholipids contents in liposomal formulation was found to cause low entrapment efficiency but it was also observed that increase in phospholipids contents along with the cholesterol amount leads to higher entrapment efficiency referred in Table 4. The liposomal formulation having a high phospholipids concentration has the higher entrapment efficiency but depends upon the cholesterol amount. It can also be seen that increasing cholesterol amount for a given amount of phospholipids increases drug entrapment efficiency. This may be due to the increased stability of phospholipids bi-layers by cholesterol [31].

In vitro Drug Release Studies of Liposomes Prepared by MEIM and TFHM Method
The release kinetics and the plots of nine formulations appeared from Fig. 2

Ex Vivo Drug Diffusion Studies of Liposomes Formed by MEIM and TFHM Methods
In ex-vivo permeability studies, Fig. 3 showed that the percentage of drug release from liposomes was increased with time. It was also observed that ratio of phospholipids and cholesterol greatly influenced the drug release profile from liposomes in both methods. Drug release pattern of the liposome formulations showed that the drug release decreased with the increase of the amount in cholesterol and phospholipids but further increase in phospholipids increased drug release [33].

Mechanism of Drug Release of Tamoxifen Loaded Liposomes
Four mathematical models such as zero order, first order, Higuchi and Korsmeyer-Peppas were used to characterize the release mechanism of tamoxifen liposomes. The kinetic constant of release models were described by K 0 , K 1 , K H and n for zero order, first order, Higuchi and Korsmeyer-Peppas respectively where highest correlation coefficient (R 2 ) was considered to express the best drug release pattern from liposomes. From Table 5 it was seen that most of the formulations (13 out of 18) followed zero order kinetics [34].
It was reported that when n is below 0.45, the Fickian diffusion phenomenon dominates whereas n between 0.45 and 0.89 is an anomalous transport often termed as first order release. Purely matrix relaxation or erosion mediated release occurs for n=0.89 (zero order kinetic). After the n value reaches 0.85 and above, the release can be characterized by class II and super class II transport, which means the drug release rate does not change over time and the release is characterized by zero order release. In this case, the drug release was dominated by the erosion and swelling of the polymer [35,36]. The release exponent 'n' was the slope of log fraction of drug release versus log time curve Fig. 2 (C). The value of diffusion exponent (n) that was shown in Table 5 proved that the formulations were following super class II transport because of the diffusion exponent (n) was more than 0.85.

Time (hour)
Additionally, for thin film hydration method, drug retarding ability increased from least to highest 1(MDT= 0.603) to TTFM-B.
Overall MDT values showed that for high amount of ol increased the retarding affinity of formulations for a certain level. After an optimum level, increase in amount of surfactant and cholesterol resulted in decreased drug retarding affinity because cholesterol starts to break the bilayer of the hich has to be controlled by the amount  (Table 4). From Table 6, it was revealed that the formulations were stable when stored at 4±2ºC, 25±2ºC and 37±2ºC at 60± 5% RH. Throughout the study period (0 to 21 days), the selected formulations from MEIM and TFHM methods were stable due to the optimum amount of phosphatidylcholine and cholesterol in liposomes which stabilized the liposomal bilayer

Physical Appearance of Liposomes under Optical Microscopy
The morphology of all the liposomal formulations were determined by optical microscope (1000X) equipped with digital camera. These photomicrographs confirmed that the liposomes were spherical vesicles Fig. 5. the test. Liposomal formulations TEI-9 and 9 were selected for this study due to having highest tamoxifen entrapment efficiency (Table 4). From Table 6, it was revealed that the when stored at 4±2ºC, ºC at 60± 5% RH. Throughout the study period (0 to 21 days), the selected formulations from MEIM and TFHM methods were stable due to the optimum amount of phosphatidylcholine and cholesterol in liposomes which stabilized the liposomal bilayer [41].

Physical Appearance of Liposomes under Optical Microscopy
The morphology of all the liposomal formulations were determined by optical microscope (1000X) equipped with digital camera. These photomicrographs confirmed that the liposomes

Comparison of Marketed Product with Liposomes
The comparison of cumulative % release of drug of an available marketed product of tamoxifen was performed with two of the formulated (TEI and TTFH-9) liposomes which showed negligible deviation of drug entrapment efficacy and optimum stability during stability test entitled in Tables 5 and 6

Comparison of Marketed Product with
The comparison of cumulative % release of drug of an available marketed product of tamoxifen was performed with two of the formulated (TEI-9 9) liposomes which showed negligible rapment efficacy and optimum stability during stability test entitled in Tables 5 and 6. Fig. 6 expressed that the prepared liposomes had a sustained release characteristics because after one hour percent release of marketed product was 85.5 whereas the formulated liposomes TEI TTFH-9 revealed 81.41% and 89.41% release respectively after 6 hours. From this observation it can be said that the prepared liposomes will release for a prolonged period of time and sustained release characteristics was obta [42].

fractional dissolution time (hour) of tamoxifen loaded liposomes a) MEIM
prepared liposomes had a sustained release characteristics because after one hour percent se of marketed product was 85.56% formulated liposomes TEI-9 and 9 revealed 81.41% and 89.41% release respectively after 6 hours. From this observation it can be said that the prepared liposomes will release for a prolonged period of time and sustained release characteristics was obtained structures of liposomes. Hence the prepared tamoxifen loaded liposomes might be a potential candidate for safe and effective controlled drug delivery system for the treatment of breast cancer. It may also reduce the risk of severe side effects of tamoxifen tablet.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
Authors are pleased to acknowledge the facilities and support provided by the Department of Pharmacy and IEERD, University of Asia Pacific.