22 journal of applied biopharmaceutics and ...24 journal of applied biopharmaceutics and...

22 Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018, 6, 22-33

E-ISSN: 2309-4435/18 © 2018 Scientific Array

Design and Optimization of Bicalutamide Loaded Liposomes for Delivery in Prostate Cancer: In vitro in vivo Evaluation

Mangesh Pralhad Patil and Pradum Pundlikrao Ige*

Department of Pharmaceutics, R C Patel Institute of Pharmaceutical Education and Research Shirpur, Dhule, Maharashtra 425405, India

Abstract: Bicalutamide (BCT) is a potent anticancer drug that has shown to be active against a broad spectrum of prostate cancer. In the present investigation, CCRD-RSM was applied successfully to the encapsulation of BCT loaded liposome. Liposomes were prepared using a thin film hydration method. The effects of processing variable that is concentration of cholesterol and soya lecithin on the dependent variables like mean vesicle size (MVS) nm, encapsulation efficiency (EE) and zeta potential (mV) was studied. Formulation were characterized by DSC, FTIR, PDI, zeta potential, solubility, percent EE, SEM, in vitro dissolution, sterility, short term stability and in vivo studies. The MVS, percent EE, zeta potential of optimized liposome formulation was found to be 138.8 nm, 98.21±9.4,-17.4 mV, respectively. The percent CDR of optimized BCT loaded liposome was found to be 92.82 at 72h. The area under curve of the optimized BCT loaded liposomes found to be 1.58 fold as compared to marketed suspension in Albino Wistar rats. In conclusion, BCT loaded liposomes could be demonstrated as a potential carrier to improve, bioavailability and circulation time of bicalutamide.

Keywords: Bicalutamide, Liposome, Thin film hydration method, Differential scanning calorimeter, Scanning electron microscope, Mean vesicle size.

1. INTRODUCTION

Nanotechnology is continuously bringing the revolutionary changes in the era of drug delivery system. Liposomes are developed as a fore frontier for lipid based drug delivery system. Nanoparticles are carrier of colloids with dimensions on the nano size. They are particularly used for cancer treatment due to of their nano size, varied composition, surface functionalization and stability provide unique opportunities to interact and target the tumors cell. Liposomes, phospholipids vesicles were discovered and soon after recognized as promising drug carriers [1].

Huge progress in the field of liposomal drug delivery has been achieved and the first generation liposomal formulations such as Doxil, Myocet and Dauno Xome have been entered the clinic. While these formulations have improved some aspects of cancer treatment, there is still ample room for improvement of liposomal cancer therapeutics, and significant advances are continuously achieved [2].

Liposome science and technology is one of the fastest growing scientific fields. This is due to several advantageous characteristics of liposomes such as ability to incorporate not only water soluble but also *Address correspondence to this author at the Department of Pharmaceutics, R.C. Patel Institute of Pharmaceutical Education & Research, Karwandnaka, Shirpur, Dhule, Maharashtra, 425405, India; Tel: +91-8668388789; Email: [email protected]; [email protected]

lipid soluble agents, specific targeting to the required site in the body and versatility in terms of fluidity, size, charge and number of lamellae. Liposomes are spherical vesicles composed of one or more phospholipids bilayers (in most cases phosphatidylcholine). Lipophilic drugs can be incorporated within the lipid bilayers while hydrophilic drugs are solubilized in the inner aqueous core. Liposomes have been extensively studied as a drug delivery system and can improve biological efficacy and reduce side effects of drugs [3-5].

Various methods have been utilized for preparation of Liposomes. There are at least fourteen major reported methods. The most commonly employed method is lipid film hydration (Thin film hydration method) (THF), reverse phase evaporation technique (REV), Rehydration-dehydration technique, Ethanol injection method Ether infusion method French press technique and detergent dialysis technique [6-8].

TFH method was selected for the preparation of Liposomes in this investigation due to nontediousness and feasible at lab and industrial scale compared to other techniques. In addition, stability point of view, the saturated phospholipids cholesterol, 1,2-diacyl-sn-glycero-3-phosphocholine (soy-hydrogenated) (HSPC) were used. Trapping efficiency is one of the prime important factors in selection of method of liposome preparation [9, 10].

The mechanism of action of Bicalutamide (BCT), N-[4-cyano-3-(trifluoromethyl) phenyl]-3-[(4-fluorophenyl)

Bicalutamide Loaded Liposomes for Delivery in Prostate Cancer Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018, Vol. 6 23

sulfonyl]-2--hydroxy-2-methylpropanamide, is a nonsteroidal antiandrogen mainly used for the treatment of prostate cancer and is orally active. It has the ability to competitively block the growth-stimulating effects of androgens on prostate cancer. Drugs with poor aqueous solubility generally show variable absorption. The low aqueous solubility of bicalutamide may be due to its polymorphism and hence the drug has been classified as a BCS class II (low solubility and high permeability). The very low solubility of bicalutamide accounts for dissolution as its rate determining step for bioavailability. Improved dissolution rate can be expected to increase oral bioavailability of the drug, resulting in reduction of dosing frequency and improved patient compliance [11-14].

In the present investigation, CCRD-RSM (Two factor three level) was applied successfully to the encapsulation of BCT loaded liposome. Liposomes were prepared using a thin film hydration method. The effects of processing variable that is concentration of cholesterol and soya lecithin on the dependent variables like mean vesicle size (MVS) nm, encapsulation efficiency (EE) and zeta potential (mV) was studied. Solubility, bioavailability and blood circulation time of bicalutamide has been enhanced through intravenous administration by liposomal delivery.

2. MATERIALS AND METHODS

2.1. Materials

Bicalutamide (BCT) obtained as a gift sample from Cipla Pvt. Ltd., Bangalore, India, the phospholipids used in the preparation of liposome such as soya lecithin purchased from Phospholipids GmbH, Germany and cholesterol purchased from Sigma–Aldrich Inc. St. Louis, MO, USA. All other chemicals used for the study were of HPLC grade. Water used in all the studies was distilled and filtered through 0.22 µm nylon filter paper before use.

2.2. Methods

2.2.1. Preparation of BCT Loaded Liposomes

In this study the most commonly employed method are used lipid film hydration also referred as thin film hydration method the most widely used method for the preparation of MLV. The different ratio of cholesterol and soya lecithin were weighed and dissolved in 10 ml of chloroform: methanol (2:1) in 250 ml round bottom flask. A thin film was formed on the inner side of round bottom flask by evaporating organic solvent under vacuum in rotary evaporator at 45-50°C. The dry lipid film was hydrated with 10 ml phosphate buffer solution (pH 7.4) containing BCT at a temperature of 60±2°C. The dispersion was left undisturbed at room temperature for 15 min to allow complete swelling of the lipid film. The vesicular suspension obtained was stored at 4°C. The schematic presentation was

Figure 1: Thin film hydration method (THF) for multilamellar vesicles.

24 Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018, Vol. 6 Patil and Ige

described in Figure1. The BCT loaded liposome dispersion was centrifuge at 25,000 rpm for 35 min at room temperature by using ultracentrifuge for the separation of nano-particulate system. Deposited particulate was redispersed in minimum amount of purified water.

2.2.2. Lyophilization of BCT Loaded Liposome

Lyophilization of an optimized Liposome dispersion was carried out with and without cryoprotectant using Virtis-Bench Top Lyophilize, Spinco Biotech Pvt. Ltd. The dispersion was pre-frozen(−75 ◦C) for 12 h and subsequently lyophilized at a temperature of−25 ◦C for 24 h followed by a secondary drying phase of 12 h at 20 ◦C. Mannitol (0.5%, w/v) was used as the cryoprotectant.

2.2.3. Mean Vesicle Size Analysis

The particle size analysis of liposome was performed using Zetasizer equipped with a 4.0 mW internal laser at a fixed angle of 90° at 25°C temp. The liposome dispersion was diluted with doubled distilled

water in a disposal polystyrene cell before analysis to get optimum 50-200 kilo counts per seconds (kcps) for measurement. The photon correlation spectroscopy yielded the mean diameter of the main population and polydispersity index as a measure for the width of the particle size distribution. Each sample was analyzed as in triplicate.

2.2.4. Zeta Potential Analysis

The zeta potential (ZP), reflecting the electric charge on the particle surface and indicating the physical stability of colloidal systems, was measured by determining the electrophoretic mobility using the Malvern Zetasizer. The measurements were performed with diluting in double-distilled water. It was measured using small cell with applying field strength 20 V/cm and the average of the zeta potential was given from 30 runs.

2.2.5. Determination of Encapsulation Efficiency and Loading Efficiency

Percent Entrapment efficiency is defined as the percentage of drug incorporated into the liposome

formulation relative to the total drug added. It specifies how much percent of drug is included in the particles and how much percent of free drug are still present in the dispersion medium.

Drug loading (DL) refers to the percentage of drug incorporated into the liposome formulation relative to the total weight of the liposomes nanoparticles.The EE% of the liposome was measered by determining the amount of entraped drug using centrifugation technique. The liposome suspension was ultra centrifuged at 5000 rpm for 15 minutes at 4°C temperature by using remi cooling centrifuge to separate the free drug. A supernant containing liposomes in suspended stage and free drug at the wall of centrifugation tube. The supernant was collected and again centrifuged at 15000 rpm at 4°C temperature for 30 minutes. A clear solution of supernant and pellets of liposomes were obtained. The pellet containing only liposomes was resuspended in distilled water until further processing. The liposomes free from unentrapped drug were soaked in 10 ml of methanol

and then sonicated for 10 min. The vesicles were broken to release the drug, which was then estimated for the drug content. The absorbance of the drug was noted at 271 nm . The entrapment efficiency and loading efficiency was calculated using following equation.

2.2.6. Surface Morphology

The surface morphology of lyophilized Liposomes and the drug–lipid melt was visualized using scanning electron microscope (Vega MV2300T/40, TS 5130 MM, Tescan) Before observation, the lyophilized nanoparticles were fixed on a double-sided sticky tape that was previously mounted on aluminum tubs and then coated with gold in an argon atmosphere. The scanning was performed at an accelerating voltage of 10 kV.

2.2.7. In vitro Drug Release

The dialysis bag diffusion technique was used as previously reported by to investigate the in vitro release of Bicalutamide loaded liposome under sink condition.


The dialysis bag was soaked in medium for 24h before use. Briefly, 4 mg of liposome powder was transferred into the dialysis bag (molecular weight cut-off 12,000 to 14,000 k Da), and dialyzed against 50 mL of pH 7.4 phosphate buffer saline solution. The medium was stirred at 50 rpm at 37±0.5°С. At designated time intervals, 5 mL dialysis medium was taken for measurement and the same volume of fresh pre-warmed medium was added. The amount of bicalutamide released from the liposome was determined spectrophotometrically at 271 nm.

2.2.8. In vivo Studies

In the present research work Male Albino Wistar rats weighing ( 200 - 250 g, ) housed in stainless-steel mesh cages in Two groups of Three rats each, under standard conditions of light illumination, relative humidity, temperature and had free access to standard laboratory food and water throughout the study maintained on pellet diet and water ad libitum were used for pharmacokinetics studies. The animal protocol was duly approved by the CPCSEA and Institutional Animal Ethics Committee of R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur (RCPIPER/IAEC/2011-12/25). First group was administered marketed BCT suspension (0.25 %) because bicalutamide was virtually insoluble in water and the second group was administered bicalutamide parenteral formulation. The amount of bicalutamide in each one of these formulations was adjusted to contain 25 mg/kg body weights. Blood samples was collected from tail vein of rat in tube containing saturated solution of di-sodium EDTA at pre-dose. During collection, blood sample has been mixed thoroughly with di-sodium EDTA solution in order to prevent blood clotting. Samples were centrifuged at 5000 rpm for 5 mins at room temperature. Separated plasma sample was transferred into pre-labeled tubes and stored at −20 ◦C until the completion of analysis. The plasma samples were diluted to 2 mL with acetonitrile, vortexed for 10 min, and then centrifuged at 8000 rpm for 20 min. Amount of 20 µL of the supernatant was injected into the HPLC column to determine the BCT plasma concentration as described above.

Determination of Pharmacokinetic Profile

Plasma was firstly mixed with 25 µL Telmisartan (internal-standard) and diluted to 2 mL with acetonitrile, was added to the same to precipitate the proteins of plasma. For 10 min vortexeing, followed by the sample was centrifuged at 8000 rpm for 20 min. From this centrifuged plasma sample, 20 µL of the supernatant

was injected into the HPLC system. The peak concentration (Cmax) and the time to reach the peak concentration (Tmax) were determined directly from the plasma concentration–time curves. The area under curve (AUC) was calculated by trapezoidal method from zero to final sampling time.

HPLC Analysis

BCT concentrations in the plasma were determined using HPLC system (Zhao et al., 2005). An Agilent HPLC system with PDA detector was used. It is composed of quaternary pump and diode array detector (Agilent 1200 Series). Chromatography was performed on a reverse-phase C18 column (Eclipsed XDB 5 µm, 4.6 mm x 150 mm,). The wavelength of this detector was set at 271 nm. Acetonitrile –Buffer containing 0.5gm potassium dihydrogen orthophosphate pH 3 maintained with ortho-phosphoric acid (60:40 v/v) was used as mobile phase. The data were acquired and processed by means of ezchrome elite software. Elution was performed isocratically at 40°C at a flow rate of 1.0 mL/min.

Data Analysis

The non-compartmental model was considered as a best suited model for calculation of the different pharmacokinetic parameters. The Cmax and Tmax were directly computed from the plasma concentration vs. time plot. The trapezoidal method was used to calculate the concentration-time curve from time 0h to 12h (AUC0→48). The Kinetica 5 software was employed for study.

Statistical Analysis

Results are expressed as the mean ± SD (Standard deviation) of at least three experiments. Statistical significance was assessed using the Student’s t -test for multiple comparisons with p < 0.05 as the minimal level of significance.

2.2.9. Sterility Test

In the present study, the formulated liposome preparation were applied for sterility testing with two media namely, alternative thioglycolate medium (ATGM) and soya bean casein digest (SBCD) medium were used to investigate the presence or absence of turbidity, foreign particles (Indian Pharmacopeia, 2010) in the formulated sterilized dosage form. The conical flask was checked at 24 h interval and the results were noted down, on eight day (after completion of 192 h). All the samples were inoculated separately in to ATGM and SBCD medium and incubated at 20-25ºC, for 8


days. The observations of sterility testing conducted with sterilized samples in two different culture media which is show Figure 7.

2.2.10. Accelerated Stability Studies

The lyophilized powder of BCT loaded liposomes optimized formulation (batch F2) was utilized for carrying out accelerated stability studies according to International Conference on Harmonization (ICH) Q1A (R2) guidelines [15] .For the products stored in refrigerator ICH guidelines suggested long term stability at 5 ± 3 ◦C and accelerated stability study at 25 ± 2 ◦C/60% RH ± 5% RH (relative humidity). Accelerated stability study was performed with the prime aim to assess the stability of SLNs at 25 ± 2 ◦C/60 ± 5% RH with respect to particle size, PDI and EE. Freshly prepared parenteral formulation was filled in 3 different amber coloured glass vials, sealed and placed in stability chamber maintained at 25 ± 2 ◦C/60 ± 5% RH for a period of total 3 months. The prepared parenteral samples subjected for stability test were analyzed with a sampling interval of 1 month for particle size, PDI and EE of the BCT loaded liposomes over 3 month period.

3. RESULTS AND DISCUSSION

3.1. Preparation of BCT Loaded Liposomes

In the present investgation Thin Film Hydration (THF) method was used which is also used in industry

for the preparation of liposomes.Chloroform and Methanol was set as a organic solvents for the extensively incorporated of drug in the lipid layer which will be hydrated with 10 ml pH 7.4 phosphate buffer solution.

3.2. Experimental Design

In the context of the CCRD-RSM design, the results of the component single-factor experiments are called the simple effects of an independent variable. Independent variables include cholesterol concentration (X1), soya lecithin concentration (Y2). Whereas mean vesicle size of Liposomes (Y1), (EE %) drug entrapment efficiency (Y2) and zeta potential (Y3) were selected as response parameter as the dependent variables. All independent variable along with their code and actual values are shown in Table 1. Three dimensional surface plots were used to ascertain the relationship between variables and responses.

3.3. Optimization Data Analysis for BCT Loaded Liposome

Response observed for all formulation batches were fited to various models using Design-Expert software. It was observed that the linear models were best-fitted for both particle size and EE. All values of R2, SD and % coefficient of variation are depicted in (Table 2). The regression equation generated for each response were given as, Results of ANOVA in (Table 2) for the

Table 1: Independent Variable along with their code, Levels and Respective Particle size, EE%, Zeta Potential of Different Batches of BCT Loaded Liposomes (n=3.These Results are Mean ± Standard Deviation

Code x1 (mg) x2 (mg) Particle size (nm) %EE Zeta potential (mV)

F1 25 125 162.33 ± 9 97.54 ± 3.9 -19.5 ±(-2.2)

F2 30 100 138.8 ± 12 98.21 ± 9.4 -17.4 ± (-6.1)

F3 25 125 162.33 ± 9 97.54 ± 3.9 -19.5 ± (-2.2)

F4 30 125 152.03 ± 2 89.9 ± 2.8 -17.6 ± (-4.6)

F5 25 100 112.1 ± 6 82.32 ± 1.22 -20.33 ± (-1.9)

F6 20 125 201.2 ± 0.7 83.43 ±7.3 -26.4 ± (-3.7)

F7 30 150 321.2 ± 23 94.9 ± 3.2 -14.4 ± (-2.8)

F8 20 150 264.3 ± 18 96.76 ± 11.3 -26.3 ± (-6.4)

F9 25 150 271.6 ± 3 98.1 ± 2.11 -20.9 ± (-3.5)

F10 25 125 162.33 ± 9 97.54 ± 3.9 -19.5 ± (-2.2)

F11 20 100 121.52 ± 1 81.21 ± 4.31 -31.1 ± (-5.1)

F12 25 125 162.33 ± 9 97.54 ± 3.9 -19.5 ± (-2.2)

F13 25 125 162.33 ± 9 97.54 ± 3.9 -19.5 ± (-2.2)

x1= cholesterol concentration in mg/ml (high level -30, low level-20); x2=soya lecithin concentration in mg/ml (high level -150, low level-100).


dependent variables demonstrated that the model was significant for both the responses and variables.

Y1 = 161.60 + 4.17A + 80.78B + 9.91AB + 16.85A2 +

32.09B2 (3)

Y2 = 93.27 + 3.60A + 4.67B - 4.72AB (4)

Y3= -19.55 + 5.57A + 1.27B - 0.20AB - 2.32A2 - 0.24B

2 (5)

3.4. RSM Plots

The three dimensional surface curves of response surface plots are important for studying the interaction patterns. Three dimensional response surface plots generated at different levels by the Design-Expert® software are presented in (Figure 2) for the studied responses, i.e. particle size and EE. From Figure 2, it can be concluded that the particle size goes on decreasing as the soya lecithin concentration (A) increases and cholesterol concentration (B) decreases. It is summarized that soya lecithin (A) and cholesterol (B) concentration has significant effect on the EE, i.e. with the increase in cholesterol concentration and decrease in soya lecithin concentration, increases the EE. Response surface plots revealed that the cholesterol and soya lecithin concentrations were statically significant.

3.4.1. Effect of Soya-Lecithin on Particle Size

The concentration of soya-lecithin which solubilized the drug in formulation has significant effect on the particle size of the BCT loaded liposome (Figure 2(a) clearly represents that, with the increase in soya-

lecithin concentration from 100 mg to 150 mg, the mean particle size of the formulation was also increased in each case. As we can say that concentration of soya-lecithin is directly proportional to the particle size. The F7, F8, and F9 batches which had the highest concentrations of soya-lecithin, relatively showed the greater particle size (in the range of 200–350 nm) than that of batches having low soya-lecithin concentration (in the range of 100–200 nm). As shown in Figure 5 (a) and Table 1.

3.4.2. Effect of soya-Lecithin on Percent EE

The effect of lipid concentration on the EE % was found to be significant (Figure 2(b). Increasing concentration of the lipid showed increasing EE % of the BCT loaded liposomes. This can be explained on the fact that as there was increase in the lipid phase, more amount of the lipid was available for the BCT to dissolve. Here, the concentration of soya lecithin is directly proportional to the Entrapment efficiency. As the highest concentration of soya-lecithin in the batches F8 and F9 shows highest entrapment efficiency.

3.4.3. Effect of cholesterol on particle size

In the present research work the increased in concentration of cholesterol decreased the particle size of the formulation. As the cholesterol shows significant effect in the BCT loaded liposomes with the concentration ranges from (20-30 mg). The batch F5 shows minimum particle size (112.1nm) due to the concentration of cholesterol. As shown in Figure 3(c) and Table 1.

Table 2: Summary of Results of Regression Analysis for Responses y1, y2 and y3 and Analysis of Variance for Particle Size, %EE and Zeta Potential

Parameter DF SS MS F p-value R2 SD %CV

Particle size (Y1)

Model 7 45224.55 9044.91 25.30 0.0002

significant 0.9476 18.91 10.27

Residual 5 2502.96 357.57 - - - - -

Total 12 47727.51 9402.48 - - - - -

%EE (Y2)

Model 3 297.61 99.20 3.89 0.0492 significant 0.5644 5.05 5.42

Residual 9 229.65 25.52 - - - - -

Total 12 527.26 124.72 - - - - -

DF, degrees of freedom; SS, sum of square; MS, mean sum of square; F, Fischer’s ratio.


3.4.4. Effect of cholesterol on Entrapment efficiency

Cholesterol shows momentous effect in relation with entrapment efficiency. As the concentration of cholesterol decrease entrapment efficiency also decreased (Inversely proportional). Total formulation batches shows entrapment efficiency in the range of (81-98 %).the optimized formulation F2 shows the highest EE% due to the concentration of cholesterol, as shown in Figure 3(d) and Table 1.

3.5. Mean Vesicle size

Vesicle size of the BCT loaded liposomes are reported in (Table 1). The mean vesicle sizes of the total formulations were found to be in the range of 112 –321 nm. An optimized formulation (F2) based on quantitative and sophisticated arrangements was selected and formulated. Particle size obtained in present study displayed the aforesaid range. This particle size range did not cause any changes in

intravenous uptake of liposomes.

3.6. Zeta potential

An electrical barrier forms on each particle surfaces due to the electric charge which results in “repulsion phenomenon’ is the zeta potential of particular formulation. Zeta potential of all batches was found to be towards negative side in the range of (−31.1 to −14.4 mV).

3.7. Encapsulation Efficiency and loading efficiency

Entrapment efficiency (EE %) of BCT loaded liposomes is mainly dependent on the nature of the drug and the lipidic phase in which the drug is encapsulated. As the BCT is a lipophilic drug and its solubility is also greater in the cholesterol, the EE obviously was found to be higher, i.e. in the range of 81–98% and drug loading was found in the range of 4-11 %. As shown in (Table 1).

A) Response surface plot showing the effect of the concentration of Cholesterol and Soya-lecithin on Mean Vesicle size

B) Response surface plot showing the effect of the concentration of Cholesterol and Soya-lecithin on Entrapment Efficiency

Figure 2: Response surface plot for the Cholesterol and Soya-lecithin concentration on particle size (a) %Entrapment efficiency (b).


3.8. Scanning Electron Microscopy

SEM photomicrograph of the pure BCT and BCT loaded liposomes is shown in Figure 5. Optimized formulation showed spherical shaped particles with an average particle size of 213 nm and the SEM images for pure BCT shows particle size 240 nm. As the internal energy used in SEM has melted the lipid which

is incorporated in the liposome formulation and damaged the morphological characterization of liposomes formulation.

3.9. In vitro drug release

In vitro drug release study of the BCT loaded liposomes showed the sustained release behavior in

Figure 3: Effect of (a) lipid concentration on particle size and (b) lipid concentration EE %.

Figure 4: Effect of (c) lipid cholesterol on particle size, and (d) EE %.


7.4 pH phosphate buffer (Figure 6). Almost liposomes formulation batches have shown the burst release with the 30% of drug release within first two hours followed by the sustained release from the BCT loaded liposomes. The presence of the free BCT in the external phase and on the surface of the nanoparticles may be the reason for this burst release. The low solubility of the BCT in aqueous phase could be the reason for the slow release of the drug from the lipid matrices after initial burst release. The increase in lipid concentration had significant effect on the BCT release which prolonged the release of the BCT from liposomes. It may be due to the equal distribution of

drug within the lipid matrix and good entrapment of BCT in lipid. Thus, with the use of the BCT loaded liposomes, it is possible to achieve the loading dose of the drug due to initial burst release and followed by maintenance dose due to the sustained release. In fact this will prevent the fluctuations in the drug plasma level.

3.10. In vivo studies

Male Albino wistar rats weighing about 200–250g were selected for the study. The six animals were housed in polypropylene cages and provided standard

Figure 5: SEM image of BCT loaded liposomes.

Figure 6: in-vitro drug profile (optimized batch F2) of BCT loaded liposome (n = 3,mean±SD).


pellet diet and water ad libitum and maintained under controlled conditions of temperature and humidity with 12 h light/ dark cycle.

The pharmacokinetic profiles of BCT after i.v. administration of BCT suspension, BCT loaded liposome formulation (25 mg/kg) are illustrated in

Figure 8 and the calculated pharmacokinetic parameters are summarized in Table 3. BCT suspension significantly prolonged the resistance of BCT in circulation system. The drug circulation time controlled to 48 h for BCT loaded liposome, which was substantially shorter than that of BCT loaded marketed suspension that was quickly removed from the

Figure 7: Sterility testing for BCT loaded liposome formulation.

Figure 8: The mean plasma concentration-time curve after a single-i.v-dose (25mg/kg) administration of the optimized formulation of BCT suspension, BCT loaded liposomes.


circulating system after administration with a plasma concentration of about 1685.72 and 516.74 ng/mL after only 1 h and 12 h, respectively.

BCT loaded liposome could extend the half-life of BCT from 30±0.115 and BCT loaded suspension could extend the half-life of BCT 180 ±0.087 (Table 4). Meanwhile, the area under the BCT concentration–time curve (AUC) of BCT suspension was increased about 1.58 folds for BCT loaded liposomes. There was an inverse relationship between the nanocarrier’s clearance and its prolonged circulation time. It has been previously observed that ultra low sized drug carriers of phospholipids may remain in circulation for a prolonged period as compared to those with a larger diameter. Based on the findings of the pharmacokinetic study, cholesterol Lipophilic shell that suppress opsonization through generating a stearic barrier preventing hydrophobic interactions of plasma opsonins with the liposome surface and thus inhibiting the uptake by RES.

3.11. Sterility Test

After performing the sterility test and the result shown that none of the tested liposomes samples

showed any germ growth, neither in the Thioglycolate nor in the soya-bean casein digest medium. The test was performed for 8 days and observed daily. As shown in Figure 7. Both the media was placed in the room temperature so no any significant effects shows in both medias. Initially both the medias was clear and after few days it was somewhat turbid due to the presence of soya lecithin in the formulation.

3.12. Accelerated stability studies

Accelerated stability studies of nano formulation were conducted by measurement of particle size, PDI, zeta potential and entrapment efficiency. There were no significant changes in particle size and PDI after three month storage. At the initial stage of the stability study of BCT loaded liposome shows mean particle size 209.4±0.91nm and at end of three months shows 213 ± 1.83 nm and PDI has 0.259±0.001 to 0.305 ± 0.027, respectively. Zeta potential of optimized batch initially was found to be -34.66±1.21 and after three month, it was found to be -35.36±1.21 mV. The Entrapment efficiency (%) of the optimized batch initially was found to be 88.36±1.20 and after three month, it was found to be 84.45 ± 1.18%.

Table 3: Pharmacokinetic Parameters of Marketed BCT Suspension, BCT Loaded Liposome

Pharmacokinetic Parameters With Units BCT Suspension BCT Loaded Liposome Nanoparticles

t1/2

h 9.3±0.12 3.7±0.22

Tmax

H 180 ±0.087 30±0.115

Cmax

ng/ml 516.72 ±108.13 1685.74±41.76

AUC 0-inf_obs

ng/ml*h 20225.51±1173.09 11041.63±157.17

MRT 0-inf_obs

H 2.2590±0.10 7.18353±0.30

Table 4: Accelerated Stability Characteristics of Freeze-Dried BCT Loaded Liposome in Terms of Mean Particle Size,

Polydispersity Index (PDI) and Entrapment Efficiency (n = 3)

Stability Parameter Test Period

0 month 1 month 2 month 3 months

MPS (nm) 209.4±0.91 204 ± 1.05 206 ± 0.89 211 ± 1.83

PDI 0.259±0.001 0.267 ± 0.035 0.291 ± 0.02 0.305 ± 0.027

Zeta potential (mV) -34.66±1.21 -34.32±1.21 -33.45±1.21 -35.36±1.21

% EE 88.36±1.20 87.56 ± 0.92 89.32 ± 1.80 84.45 ± 1.18


CONCLUSION

The factorial design will improve our understanding of the process for manufacturing of liposome using thin film hydration method. TFH method was selected for the preparation of Liposomes due to non-tediousness and feasible at lab scale. The saturated phospholipids cholesterol, 1,2-diacyl-sn-glycero-3-phosphocholine (soy-hydrogenated) (HSPC) were used in the preparation of liposomes. Entrapment efficiency is one of the prime important factors in selection of method of liposome preparation. Formulation variables used in liposome formulation affect on the mean vesicle size, zeta potential, encapsulation efficiency in addition to variation in this attributes. The optimization was done on the basis of minimization of particle size, zeta potential and maximization of encapsulation efficiency, drug loading significantly. Bicalutamide was successfully incorporated into liposomes by thin film hydration technique with high entrapment efficiency due to its high lipophilicity. Thus, liposomes can be demonstrated as a potential carrier to improve solubility, bioavailability and blood circulation time of BCT with higher entrapment efficiency. Liposomes are unique drug delivery system which can be administrated orally, parenterally and topically and thus can be used in controlling and targeting drug delivery.

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Received on 13-12-2018 Accepted on 22-12-2018 Published on 26-12-2018 DOI: http://dx.doi.org/10.20941/2309-4435.2018.06.4

© 2018 Patil and Ige; Licensee Scientific Array. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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