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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)
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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.
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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.
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Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018,
Vol. 6 25
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
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26 Journal of Applied Biopharmaceutics and Pharmacokinetics,
2018, Vol. 6 Patil and Ige
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).
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Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018,
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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.
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28 Journal of Applied Biopharmaceutics and Pharmacokinetics,
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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).
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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 %.
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30 Journal of Applied Biopharmaceutics and Pharmacokinetics,
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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).
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Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018,
Vol. 6 31
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.
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32 Journal of Applied Biopharmaceutics and Pharmacokinetics,
2018, Vol. 6 Patil and Ige
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
-
Bicalutamide Loaded Liposomes for Delivery in Prostate Cancer
Journal of Applied Biopharmaceutics and Pharmacokinetics, 2018,
Vol. 6 33
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
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