PEGylated liposomes of anastrozole for long-term ...

http://informahealthcare.com/lpr ISSN: 0898-2104 (print), 1532-2394 (electronic) J Liposome Res, Early Online: 1–19 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2015.1029493

PEGylated liposomes of anastrozole for long-term treatment of breast cancer: in vitro and in vivo evaluation Gopal Venkatesh Shavi1,2, Meka Sreenivasa Reddy2, Ramesh Raghavendra1, Usha Yogendra Nayak2, Averineni Ranjith Kumar3, Praful Balavant Deshpande2, Nayanabhirama Udupa2, Gautam Behl4, Vivek Dave2, and Kriti Kushwaha5

Journal of Liposome Research Downloaded from informahealthcare.com by 193.1.184.244 on 04/14/15 For personal use only.

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South Eastern Applied Material Research Centre (SEAM), WIT, Waterford, Ireland, 2Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India, 3Tranzderm Solutions, Brookings, SD, USA, 4Pharmaceutical and Molecular Biotechnology Research Centre (PMBRC), WIT, Waterford, Ireland, UK, and 5Department of Pharmaceutics, Banasthali University, Banasthali, India Abstract

Keywords

The aim of present study was to develop conventional and PEGylated (long circulating), liposomes containing anastrozole (ANS) for effective treatment of breast cancer. ANS is a thirdgeneration non-steroidal aromatase inhibitor of the triazole class used for the treatment of advanced and late-stage breast cancer in post-menopausal women. Under such disease conditions the median duration of therapy should be prolonged until tumor regression ends (431 months). Liposomes were prepared by the thin film hydration method by using ANS and various lipids such as soyaphosphatidyl choline, cholesterol and methoxy polyethylene glycol distearoyl ethanolamine in different concentration ratios and evaluated for physical characteristics, in vitro drug release and stability. Optimized formulations of liposome were studied for in vitro cytotoxic activity against the BT-549 and MCF-7 cell lines and in vivo behavior in Wistar rats. Preformulation studies, both Fourier transform infrared study and differential scanning calorimetry analysis showed no interaction between the drug and the excipients used in the formulations. The optimized formulations AL-07 and AL-09 liposomes showed encapsulation efficiencies in the range 65.12 ± 1.05% to 69.85 ± 3.2% with desired mean particle size distribution of 101.1 ± 5.9 and 120.2 ± 2.8 nm and zeta potentials of 43.7 ± 4.7 and 62.9 ± 3.5 mV. All the optimized formulations followed Higuchi-matrix release kinetics and when plotted in accordance with the Korsemeyer–Peppas method, the n-value 0.55n51.0 suggests an anomalous (non-Fickian) transport. Likewise, the PEGylated liposomes showed greater tumor growth inhibition on BT-549 and MCF-7 cell lines from in vitro cytotoxicity studies (p50.05). Pharmacokinetic study of conventional and PEGylated liposomes in Wistar rats demonstrated a 3.33- and 20.28-fold increase in AUC(0–1) values when compared to pure drug (p50.001). Among the formulations, PEGylated liposomes showed encouraging results by way of their long circulation and sustained delivery properties for effective treatment of breast cancer.

Anastrozole, breast cancer, chemotherapy, cytotoxic, PEGylated liposomes

Introduction Breast cancer is a troublesome disease that strikes women and is the second leading cause of death, in women with a worldwide yearly estimate of more than 1.1 million new cases of invasive breast cancer and more than 4 00 000 deaths per year in keeping with the high incidence (Nabholtz, 2006, 2008; Nabholtz et al., 2000). It is projected that worldwide more than 508 000 women died in 2011 due to breast cancer. Even though breast cancer is consideration to be a disease of the developed countries, virtually 50% of breast cancer cases and 58% of deaths occur in less developed countries. Belgium had the highest rate of breast cancer, followed by Denmark and France. The highest incidence of breast cancer was in Northern America and Address for correspondence: Gopal Venkatesh Shavi, Formulation Scientist, South Eastern Applied Material Research Centre (SEAM), WIT, Waterford, Ireland. Tel: +353 51834159. E-mail: [email protected]

History Received 21 October 2014 Revised 24 January 2015 Accepted 11 March 2015 Published online 8 April 2015

Oceania; and the lowest incidence in Asia and Africa. According to the World Cancer Research Fund global network, survival rates ranged from 90$ to less than 50%, depending on the features of the tumor, its size and spread, and the obtain ability of treatment. Five years survival rates having breast cancer are more than 80% in North America, Sweden, Japan, Finland and Australia compared with less than 60% in Brazil and Slovakia and less than 40% in Algeria. The low survival rate in medium and small income countries can be explained mainly by a lack of early detection systems resulting in a high percentage of women presenting with latestage breast cancer, as well as by a lack of adequate diagnosis and treatment facilities. Incidence rates vary greatly worldwide from 20 per 100 000 women in Eastern Africa to 90 new cases per 100 000 women in Western Europe. In most of the developing regions the incidence rates are below 40 per 100 000 (Coleman et al., 2008; WHO, 2008). The lowest incidence rates are found in most African countries but here breast cancer incidence rates are also escalating. In contrast,


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breast cancer mortality rates in these two regions are almost indistinguishable at about 15 per 100 000, which evidently points to a later diagnosis and much lesser survival in eastern Africa. An imperative need in cancer control today is to develop effective and affordable approaches to the early detection, diagnosis and the treatment of breast cancer among women living in less developed countries. The treatment of breast cancer has included efforts to decrease estrogen levels by the use of anti-estrogens by the inhibition of the enzyme, aromatase that catalyzes the conversion of the androgens, androstenedione and testosterone, to estrogens (estrone and estradiol), the major route of estrogen synthesis in post-menopausal women and progestational agents (Nicolini et al., 2006; Thomas et al., 2009). Third-generation type-II non-steroidal aromatase inhibitors (AIs) [e.g. anastrozole (ANS) and letrozole] are replacing tamoxifen as the first-line treatment-of-choice and are more potent for the treatment of breast cancer based on the diseased conditions of the patients (Wiseman & Adkins, 1998). ANS, chemically known as 2,20-(5-((1H-1,2,4-triazole-1yl)methyl)-1,3-phenylene)-bis(2-methylpropanenitrile), is a potent third-generation non-steroidal AI of the triazole class, approved by the United States of Food and Drug Administration (USFDA) for the treatment of advanced breast cancer in post-menopausal women (http://www.accessdata.fda.gov/drugsatfda). ANS (ArimidexÕ ) administered as a 1-mg tablet by oral route once daily. For patients with advanced breast cancer and for adjuvant treatment of early breast cancer in post-menopausal women, the median duration of therapy should be prolonged (Gopal et al., 2011). The purpose of this research was to design a sustained release of the anticancer agent. One of the technological resources used to improve the performance of drugs at the site of action is the use of therapeutic systems prepared using a biodegradable lipid carrier system. Liposomes or lipid vesicles are colloidal particles composed of (phospho) lipid molecules as the major constituent in formation of enclosed lipid bilayers or lipid– drug sheet-disk complexes. Lipid vesicles have shown an increasing importance in the development of sustained drug delivery system. Although the lipid constituent can vary, many formulations use synthetic products of natural phospholipids, mainly phosphatidylcholine (Kim et al., 2006; Park, 2002). Liposomes stability in storage and disposition in vivo (in particular, plasma clearance) are two of the most important parameters for parenteral preparations of liposome-based therapeutics (Jain & Jain, 1997; Tianshun & Rodney, 2001). The major drawbacks of the liposomal formulation are its rapid clearance from blood due to the adsorption of plasma protein to the phospholipid membrane of the liposomes, which triggers the recognition and uptake of the liposomes by the mononuclear phagocytic system (MPS). When the surface of the liposomes is modified with flexible hydrophilic polymers such as polyethylene (glycol-PEG), the uptake by MPS is retarded (Schnyer & Huwyler, 2005; Torchilin & Trubetskoy, 1995). PEGylation is generally described as the molecular attachment of polyethylene glycols (PEGs) with different molecular weights to active drug molecules or surface treatment of drug-bearing particles with PEGs and is one of the most promising and extensively studied strategies for improving the pharmacokinetic behavior

J Liposome Res, Early Online: 1–19

of therapeutic drugs by increasing their circulation time in the body. In this article, a simple thin film hydration method was used for preparing conventional and PEGylated liposomes (Crommelin et al., 1997) through a combination of high pressure homogenizer and extrusion through polycarbonate membrane filters to form uniform size vesicles (monodisperse) for intravenous administration to improve their pharmacokinetic profiles, conventional and PEGylated liposome’s by prolonging their circulation time in the blood. Further optimized formulations were subjected to Fourier transform infrared (FTIR) study, differential scanning calorimetry (DSC) analysis, particle size distribution, zeta potential, in vitro cytotoxicity, stability studies and in vivo behavior in rats.

Materials and methods Materials ANS (99.5% purity) was obtained as a gift sample from Sun Pharmaceuticals PVT, Ltd., Baroda, India. Soyaphosphatidyl choline (SPC100), cholesterol (CH) and methoxy polyethylene glycol distearoyl ethanolamine (MPEG-DSPE2000) were purchased from Sigma Aldrich Chemie, Bangalore, Karnataka, India. Acetonitrile (HPLC grade) was purchased from Merck Specialties Pvt., Ltd., Mumbai, India. Monomeric column C18 (250 mm 4.6 mm, 5 mm) was purchased from Gracevydac, Frankfurt, Germany. All other chemicals used were of analytical grade. Millipore water (Millipore, Bedford, MA) was used throughout the study. Formulation of conventional and PEGylated liposomes The thin film hydration technique as described by Bangham & Horne (1964) and Bangham et al. (1965) was followed for the preparation of conventional and PEGylated liposomes. The lipids used were SPC100, CH and MPEG-DSPE2000. For preparation of liposomes, accurately weighed amounts of lipids with different lipid molar ratios as shown in Table 1 and drug (5 mg) was added into a 100-mL round bottom flask (RBF), then dissolved in 5 mL of chloroform and was attached to rotary evaporator (Labortechnik AG, Buchi, Switzerland). The water bath temperature was maintained at 40 C, with a rotation speed of 50 rpm and the organic solvent was evaporated under reduced pressure (20 mbar) by placing the round bottomed flask on a rotary evaporator. The process was allowed to continue for an additional 30 min until all the solvent is evaporated and a lipid film was formed on the walls of the flask (Azmin et al., 1985). Later vacuum was released; RBF was kept in vacuum desiccators (12 h) for complete evaporation of residual traces of chloroform and to form a thin lipid dry film. The thin film was hydrated with 5 mL of phosphate buffered saline (PBS) (pH 7.4) with or without 3.0% v/v Tween-80 by rotating the flask for 60 min with a rotation speed of 100 rpm at a bath temperature of 40 C until the lipid film was completely hydrated to liposome dispersion. Then, liposome dispersion formed was passed through a high pressure homogenizer (Emulsiflex-C3, Avestin, Mannheim, Germany) at a pressure of 10 000 psi for five cycles, sequentially extruded through 1.2, 0.4 and 0.2 lm pore size membrane filters using minisartÕ units to get small and

PEGylated liposomes of anastrozole

DOI: 10.3109/08982104.2015.1029493

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Table 1. Formulation composition for the prepared liposomes. Batch No.

AL-01

AL-02

AL-03

AL-04

AL-05

AL-06

AL-07

AL-08

AL-09

AL-10

Lipids molar ratio. ANS (mg) SPC (mg) Cholesterol (mg) MPEG-DSPE2000 (mg) Chloroform (mL) Tween-80 (% v/v) Trehalose (mg) Hydration volume (mL)

(4:1) 5.0 80.27 10.0 – 5.0 – – 5.0

(6:1) 5.0 120.4 10.0 – 5.0 – – 5.0

(8:1) 5.0 160.53 10.0 – 5.0 – – 5.0

(9:1) 5.0 180.6 10.0 – 5.0 – – 5.0

(9:2) 5.0 140.46 20.0 – 5.0 – – 5.0

(9:3) 5.0 120.0 30.0 – 5.0 – – 5.0

(9:1) 5.0 180.6 10.0 – 5.0 3.0 50.0 5.0

(9:1:0.25) 5.0 180.6 10.0 25 5.0 3.0 50.0 5.0

(9:1:0.5) 5.0 180.6 10.0 50 5.0 3.0 50.0 5.0

(9:1:1) 5.0 180.6 10.0 100 5.0 3.0 50.0 5.0

more uniform sized distribution of liposomes. The suspension was allowed to stand undisturbed for about 2 h at room temperature under nitrogen gas to allow the liposomes to anneal and stabilize. The liposome dispersion was stored in a refrigerator at 4 C until being used. After the optimization of conventional liposomes, PEGylated liposome was formulated by varying the amount of MPEG-DSPE2000 with the same soyaphosphatidyl choline–CH (9:1) ratio, hydration volume and time. For separation of liposomes like, for hydrophobic drugs, the centrifugation method is widely used. Free ANS was removed from the liposome suspensions, initially by centrifuging at 2000 rpm for 10 min, after which the supernatant liposomal dispersion was centrifuged at 57 438g using a refrigerated centrifuge (Sigma 3K30, Germany) at 4 C for 60 min to precipitate the liposomes (Elron-Gross et al., 2009; Yang et al., 2007a,b,c). Complete precipitation of the liposomes was confirmed by observing the absence of particles in the supernatant, then the supernatant was separated and the liposome pellet washed three times with millipore water. The pellet was then suspended in millipore water containing trehalose (50 mg/mL) and freeze-dried. Liposome dispersions were freeze-dried using trehalose (50 mg/mL) in millipore water as cryoprotectant. Liposome samples were stored in glass vials rapidly frozen and maintained at 60 C for 12 h (freezing), drying was performed at 48 C for approximately 48 h, under vacuum pressure of 50 mMtorr using freeze drying process, LabtechÕ Freeze Dryer, LFD-5508, Daihan Labtech Co. Ltd., Korea. After effective drying freeze-dried sample was reconstituted to original volume with PBS (pH 7.4) and evaluated for any change in their particle size, polydispersity index (PDI) and zeta potential. Characterization of liposomes Percentage encapsulation efficiency of conventional and PEGylated liposomes The encapsulation efficiency (EE) is defined as the ratio of the amount of the ANS encapsulated in liposome to that of the total ANS in the liposomal dispersion (Kaiser et al., 2003; Musteata & Pawliszyn, 2006). After separation of liposome pellets (entrapped drug) and removing the supernatant by aspiration, a 100 mL of entrapped drug liposomes (liposome pellets) was taken in 2 mL Eppendorf centrifuge tube and 400 mL triton-X (10% v/v) was added followed by 200 mL methanol and 300 mL mobile phase then the volume was made up to 1 mL, centrifuged at 10 000 rpm for 5 min and clear supernatant was injected to HPLC system to determine the

amount of drug in liposomes. An aliquot (100 mL each) of the liposome suspension was also dissolved as described above to determine the total amount of ANS in the liposome suspension, after which the EE was calculated from the following equation: ð%ÞEE ¼

Amount of ANS in liposomal pellet ðmgÞ 100: Amount of ANS in liposomal dispersion ðmgÞ

An aliquot (10 mg each) of the freeze-dried liposome powder was dissolved with the same mixed solvent (1 mL) to determine the content of ANS in the freeze-dried liposome powder using the following equation after appropriate dilution with the same solvent mixture as described above: Content ¼

Amount of ANS in freeze dried liposomes ðmgÞ : Amount of freeze dried liposomes ðmgÞ

Particle size analysis and zeta potential The liposomes mean diameter and particle size distribution were determined using dynamic light scattering (DLS) technique with a ZetasizerNano ZS (Malvern Instruments, Worcestershire, UK). Liposomes were diluted (1 in 10) with PBS buffer (pH 7.4) and all measurements were carried out at 25 C, assuming a medium viscosity of 1.0200 and medium refractive index (RI) of 1.335. The values of viscosity and RI are available in the software of the instrument, having list of media to select. Therefore, when analyzing the samples viscosity and RI related to water (equivalent to PBS) was selected and used as medium of dispersion to analyze particle size, PDI and zeta potential measurements. The results are presented as an average diameter of the liposomes suspension (Z-average mean) with the PDI. The particle size distribution was characterized using PDI, which is a measure for the width of the size distribution. Measurement of the zeta potential of samples in the ZetasizerNano ZS (Malvern Instruments) is performed using a combination of laser Doppler velocimetry and phase analysis light scattering (Patel et al., 2013). In this technique, a voltage is applied across a pair of electrodes at either end of a cell containing the particle dispersion. Charged particles are attracted to the oppositely charged electrode and their velocity is measured and expressed in unit field strength as their electrophoretic mobility. The liposome samples were diluted (1 in 10) with PBS (pH 7.4). Measurements were carried out at 25 C. Particles with zeta potentials more positive than +30 mV or more negative than 30 mV are normally considered electrophoretically stable (Alexopoulou et al., 2006; Shah & Misra, 2004).

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FTIR spectroscopy Infrared spectroscopy of liposomes of ANS, SPC, CH, trehalose, MPEG-DSPE2000, physical mixtures (1:1) were recorded by using a Shimadzu FTIR 8300 Spectrophotometer and the spectra were recorded in the region of 4000– 400 cm1. The procedure consisted of dispersing a sample (ANS and liposome) in KBr (200–400 mg) and compressing into disks by applying a pressure of 5 tons for 5 min in a hydraulic press. The pellet was placed in the light path and the spectrum was obtained.


dispersed in fresh medium and it was then transferred to the vial and the study was continued. The amount of ANS released in each sample, suitably diluted with mobile phase (acetonitrile–10 mM phosphate buffer, pH 3.0, 60:40) was determined using a calibration curve by using HPLC (Shimadzu LC 2010HT series) equipped with a UV detector and absorbance measured at 210 nm. The experiments were carried out in duplicate and expressed as the mean ± SD. Drug release data were normalized by converting the drug concentrations in solution to a percentage of cumulative drug release and are shown graphically.


DSC analysis DSC analysis of ANS, SPC, CH, trehalose, MPEG-DSPE2000 and trehalose was performed by using a DSC-60. The instrument is comprised a calorimeter (DSC 60), a flow controller (FCL 60), a thermal analyzer (TA 60) and operating software TA 60 from Shimadzu, Bangalore, India. The samples (ANS and liposomes) were placed in aluminum pan and were crimped, followed by heating under a nitrogen flow (30 mL/min) at a scanning rate of 5 C/min from 25 to 200 C. An aluminum pan containing the same quantity of indium was used as reference. The heat flows as a function of temperature were measured for both the drug and drugexcipient mixture. Negative staining transmission electron microscopy Negative staining transmission electron microscopy (NSTEM) was used to assess the size and surface morphology of the liposomes. NSTEM visualizes relatively electron transparent liposomes as bright areas against a dark background. The optimized liposomal dispersions were diluted 10 times with millipore water in Eppendorf tubes with a 200 lL fixed volume pipette (Eppendorf, Hamburg, Germany). The 400-mesh copper grid, which had previously been coated with formvar and carbon immersed face down in the droplet for 2 min and the surplus was removed by filter paper. Then, the grid was incubated in 1% (w/v) uranyl acetate for 1–2 min and rinsed with millipore water to wash off the excess stain and dried at room temperature. The prepared sample was observed in a Morgagni 268 D transmission electron microscope (FEI, Eindhoven, The Netherlands) operated at 80 kV at a final magnification of 120 000. The magnification of the microscope was calibrated with standard latex spheres (Chen et al., 2009; Yang et al., 2009). Three grids were prepared for each sample and the grid openings were randomly selected and viewed. In vitro drug release For in vitro drug release studies of both conventional (AL-01 to AL-04 and AL-07) and PEGylated (AL-08 to AL-10) liposomes were carried out by the vial method (Schliecker et al., 2004). Here, 10 mL of PBS pH 7.4 with 0.01% sodium azide (preservative) was taken in a 30-mL vial. Liposomes containing single dose equivalent to ANS was added to the vial and kept on water bath shaker at 37 C. Samples (1 mL) were withdrawn at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 and 168 h for both conventional and PEGylated liposomes and were centrifuged at 10 000 rpm for 5 min to get pellets at the bottom. Each time the pellet after centrifugation was

In vitro cytotoxicity studies in human breast cancer cells Anticancer potential of developed optimized liposomal formulations (AL-07 and AL-09) was evaluated in human breast cancer cell lines in vitro. Cytotoxic potential of these liposomes was studied in the cell lines and compared against the pure drug. The effects of pure drug and optimized formulations (AL-07 and AL-09) on the viability of BT-549 and MCF-7 (breast cancer cell lines) were determined by 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). Cells were cultured in Dulbecco’s modified essential medium with 10% fetal bovine serum containing penicillin (100 units/mL) and streptomycin (100 lg/mL). In all experiments, cells were maintained at 37 C in a humidified 5% CO2 incubator. Briefly, 8 104 cells per well were plated in 96-well plates and incubated for 24 h. Pure drug and optimized formulations (AL-07 and AL-09) solution were freshly prepared in 1 PBS and filtered through 0.22 mm syringe filters. Serial dilution of the stock solutions was performed in growth media to get final concentrations of 0.01, 0.1, 1.0, 10 mM, respectively. Then, 100 lL solutions of pure drug and formulations at specified concentrations were added to the growth medium (in triplicates) and further incubated for another 48, 72 and 168 h, respectively, under the same conditions separately. To evaluate cell survival, 20 lL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h. At the end of incubation period, the medium containing MTT was gently replaced by 200 lL dimethyl sulfoxide to dissolve formazan crystals and the absorbance was measured by a microtiter plate reader (Biotek ELX800–MS) at 540 nm with a reference wavelength of 630 nm. The absorbance of test (treated cells) and the control (untreated cells) was used for the determination of the percentage cell viability. Cell survival in control cells was assumed to be 100%. IC50 values, concentration of the pure drug or formulations (AL-07 and AL-09), which reduce the cell viability to 50% were determined. Percentage of cell viability was calculated by the following equation: % Cell viability ¼

O:Dof test 100: O:Dof control

Pharmacokinetic studies of optimized formulations The pharmacokinetic studies were carried out in Wistar rats, weighing 150–200 g obtained from the Central Animal House (IAEC/KMC/18/2008–2009) at Manipal University in Manipal, India. They were housed and maintained under controlled conditions of temperature (25 C/50% RH) for 12 h


DOI: 10.3109/08982104.2015.1029493

light–dark cycle in polypropylene cages filled with sterile paddy husk. They were fed with balanced diet (food) and water ad libitum. The study protocol was approved by the Institutional Ethical Committee, Kasturba Medical College, Manipal University, Manipal. The overnight fasted animals were divided into three groups (n ¼ 6) and treated intravenously with a dose of 1.0 mg/kg, which was reconstituted using water for injection (WFI) as below. Group I: Pure ANS (active ingredient). Group II: Conventional liposomes (AL-07). Group III: PEGylated liposomes (AL-09). The blood samples were collected at predetermined intervals of 0.08, 0.16, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h post-dose into heparinized tubes from the orbital sinus. The blood samples were immediately centrifuged by using centrifugation (Remi Equipments Ltd., Mumbai, India) at 10 000 rpm for 5 min and the plasma was stored at 70 C until analysis. The pharmacokinetic parameters, described in Table 4 (pharmacokinetic parameters of optimized formulations) was calculated using a non-compartment model analysis, with PK Solution v.2.0Õ software, Montrose, CO. Statistical analysis The data obtained from the pharmacokinetic determinations were expressed as mean ± SD. One-way analysis of variance followed by Tukey post hoc test was used for comparison between the multiple groups. A p value of 0.001 was denoted as indicative of a statistically significant difference. The GraphPad PrismÕ Version 4.0 Software (GraphPad Software, Inc., La Jolla, CA) was used to analyze the results. A two-tailed unpaired Student’s t-test was used to compare difference between two different groups. Stability studies A stability study was performed to investigate the leaking of drug from the liposomes during storage. The formulations were stored in hermetically sealed clear glass vials. The stability studies are carried out at accelerated storage condition, 25 ± 2 C/60 ± 5% RH, as per ICH/USFDA guidelines ‘‘Q1A (R2) 2003, Stability Testing of New Drug Substances and Products’’ to show the stability of the formulations at long-term storage condition, at 5 ± 3 C. From the study, two optimized formulations, conventional (AL-07) and PEGylated (AL-09) liposomes (n ¼ 3) were hermetically sealed in 5 mL clear glass vials and stored at 5 ± 3 C with an accelerated storage condition of 25 ± 2 C/ 60 ± 5% RH for a period of 180 days (Kim et al., 2006; Zhang et al., 2005). As a function of time, after 7, 15, 30, 60, 90 and 180 days of storage period, the samples were tested for following attributes at each temperature, 5 ± 3 and 25 C. Change in particle size, PDI and zeta potential. Change in the EE.

Results and discussion Formulation development As biodegradable and essentially non-toxic vesicles, liposomes have been used as aseptic delivery vehicles for


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several anti-cancer agents e.g. DoxilÕ , caylex: PEGylated liposomal loaded with doxorubicin (Johnson and Johnson, New Brunswick, NJ), Marqib-vincristine sulfate liposomes injection – vincristine-encapsulated liposomes in a lipid bilayer of sphingomyelin by Hana Biosciences, DaunoXome (Daunorubicin citrate-liposome injection by NeXstar Pharmaceuticals, DepocytTM: Liposomal cytarabine or liposomal Ara-C are antimetabolite cytarabine, encapsulated into multivesicular lipid-based particles (Enzon Pharmaceuticals) etc. In this study, conventional and PEGylated liposomes were prepared by the thin film hydration method (simple and reproducible) and evaluated for physicochemical and in vivo studies. Once after hydration of thin lipid layer, the liposomes were passed through a high pressure homogenizer at a pressure of 10 000 psi for five cycles (optimized condition), kept constant for all formulations to give uniform populations of liposome dispersions which were analyzed for particle size distribution. Hydration volume, centrifugation speed and time for separation of entrapped liposomes were optimized and thereafter kept constant for all formulations. Further an increase in the hydration time may affect the stability of the ANS therefore hydration time was optimized for complete hydration of thin lipid film in the preparation of liposomes and found to be 60 min. One of the major challenges of freeze drying the liposomes is the preservation of the structural integrity of the liposome during freeze drying/reconstitution process. Freeze dryer parameters, i.e. for 48 h, were pre-fixed based upon research literatures and the background experience of the formulation. With variation in freeze drying time, agglomerated gel and/or fluffy mass was observed, might be due to insufficient drying. From the trial batches with or without drug, the freeze drying parameters were optimized and further utilized for formulations. Sugars have been reported to act as cryoprotective agents during the freeze drying/reconstitution of liposomes by preventing vesicle fusion and helping retention of the encapsulated compounds within the liposomes. Hence, in this study, trehalose as cryoprotectant was found to be effective at a concentration of 50 mg/mL (optimized), was used for freeze drying process and kept constant for both the conventional (AL-07) and PEGylated (AL-08 to AL-10) liposomes. This concentration was used based upon performing varying concentration trials, i.e. 25 and 100 mg/mL. At 25 mg/mL agglomerates were observed with insufficient reconstitution, whereas 100 mg/mL was increasing bulk volume for 1 mL of dispersant. The results of varying particle size distribution, PDI, EE and zeta potential value upon using low and high concentration of cryoprotectant are depicted in Table 2. Hence, 50 mg/mL concentration of trehalose was used, from which the liposomes were found to be of acceptable physicochemical properties. The freeze drying cycle was also optimized at conditions as described above to enhance their physicochemical stability during storage. After the freeze drying process, the amount of drug present in freeze dried powder was analyzed for further studies (cell lines, pharmacokinetic and stability studies). The initial optimization was carried out in conventional liposomes, by increasing the concentration of SPC concentration, followed by varying the concentration of CH, i.e. AL-01 to AL-06. Based on the previous reports, among the surfactants available for clinical use, 3.0% v/v

0.332 ± 0.02 Completely dried, slight yellowish powder White to slight yellowish suspension 0.521 ± 0.04 Agglomerated white to slight yellowish mass Agglomerates still remaining

Tween-80 (optimized concentration) was added to increase the solubility of the drug and to enhance the EE and the stability of the liposomes for a lipid molar ratios of 9:1 (SPC– CH) and 9:1:0.5 (SPC–CH–MPEG-DSPE2000). Further increases in the Tween-80 concentration resulted in significant liposomes destruction. The results of varying particle size distribution, PDI, EE and zeta potential value upon using low and high concentration of surfactant are depicted in Table 2. To the optimized conventional liposomes (AL-07) different amounts of MPEG-DSPE2000 were added and the abovementioned physicochemical parameters were used to optimize the process parameters for PEGylated liposome production. The process followed was simple and yielded liposomes with minimal batch-to-batch and intra-batch variations indicating reproducibility. Characterization of liposomes Percentage EE

All values are mean ± SD (n ¼ 3).

Re-dispersion properties

0.332 ± 0.02 Completely dried, white to slight yellowish powder White to slight yellowish suspension 0.654 ± 0.1 Agglomerated white to slight yellowish gel mass Not re-dispersible due to gel mass

0.410 ± 0.05 Completely dried, white to slight yellowish powder Thick suspension due to bulk volume

0.852 ± 0.4 Agglomerated white to slight yellowish gel mass Not re-dispersible due to gel mass

80.95 ± 1.4 164.43 ± 1.9 59.5 ± 2.4 69.85 ± 3.2 120.2 ± 2.8 62.9 ± 3.5 46.1 ± 1.9 325.46 ± 3.9 62.9 ± 3.5 69.85 ± 3.2 120.2 ± 2.8 62.9 ± 3.5 67.15 ± 2.2 225.2 ± 4.1 55.9 ± 2.5

% EE PSD (nm; mean ± SD) Zeta potential (mV; Mean ± SD) PDI Physical appearance after freeze drying

50 mg/mL 25 mg/mL

65.14 ± 1.5 153 ± 3.2 49.8 ± 2.5

8.9 ± 2.9 615.85 ± 2.3 38.9 ± 3.6

4.0% 3.0%

0.452 ± 0.06 Completely dried, white to slight yellowish powder White to slight yellow suspension with liposomes destruction


Parameters

Concentration of cryoprotectant

100 mg/mL

1.0%

2.0%

Concentration of surfactant

G. V. Shavi et al.

Table 2. Effect of trehalose and Tween-80 concentrations on percentage EE, particle size distribution (PSD), PDI and zeta potential (n ¼ 3).


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Encapsulation of drug in liposomes is dependent on numerous factors one of which is the lipid ratio, i.e. ratio of CH and phospholipid used in the preparation. Another factor which can affect the encapsulation is the ratio of drug to total lipids. The different formulations of ANS loaded in conventional and PEGylated liposomes are summarized in Figure 1 and Table 3. We determined the total amount of encapsulate found in the liposome solution by measuring the incorporated drug present in the liposome pellets, after separation of liposomes by centrifugation. The formulations, AL-01 to AL-04 with increased SPC content EE were found to be in the range values, from 36.93 ± 1.57 to 55.26 ± 1.84 and formulations AL-05 and AL-06 with increased amount of CH showed value of 59.1 ± 2.14% and 50.30 ± 2.04%, respectively. Further AL-07 with 3.0% v/v Tween-80 and AL-08 to AL10 formulations (increased amounts of MPEG-DSPE2000) showed EE values of 65.12 ± 1.05%, 66.38 ± 1.2%, 69.85 ± 3.2% and 67.16 ± 1.04%, respectively. Among all the formulations, AL-07 and AL-09 showed maximum EEs. From the results obtained we found that as the amount of SPC and MPEG-DSPE2000 increased in the formulation of liposomes, thereby improving EE (Atyabi et al., 2009). Tween-80 appeared to have increased the content of ANS in the inner water phase due to its solubilizing effect and to have decreased the leakage of ANS from the lipid bilayer, similar to results were reported by Yang et al. (2007a,b,c). As the drug is poorly soluble, a non-ionic surfactant, Tween-80 was used to enhance solubility and intrinsic dissolution rate. It aids solubility by wetting and solubilization of hydrophobic drugs. The mechanism is presumed that, during hydration, the Tween-80 may permeate between drug and lipid junction and thereby facilitate to enhance the solubilization of drug. For further increase in MPEG-DSPE2000 in AL-10 formulation there was no significant difference in the amount of drug encapsulated. This may be due to an increase in binding of ANS to the lipids. Previous reports have shown that CH use in formulations is known to condense the packing of phospholipid bilayers (Hathout et al., 2007; Jain et al., 2005; Sezer et al., 2004). When the amount of CH is increased, EE also decreased (Betageri & Parsons, 1992). The lipophilic nature of ANS molecules may compete with CH for the hydrophobic



DOI: 10.3109/08982104.2015.1029493

7

Figure 1. Percentage EE of ANS-loaded conventional and PEGylated liposomes.

Table 3. Results of the physico-chemical characterization parameters for different formulations (n ¼ 3). Batch Nos. Parameters

AL-01

AL-02

AL-03

Particle 164.44 ± 4.9 173.8 ± 8.9 183.0 ± 7.1 size (nm) Zeta potential 26.46 ± 2.9 27.79 ± 4.8 32.75 ± 6.7 (mV) PDI 0.671 ± 0.06 0.461 ± 0.04 0.429 ± 0.03 EE % 36.93 ± 1.57 41.09 ± 1.96 48.14 ± 2.43

AL-04

AL-05

AL-06

AL-07

AL-08

AL-09

AL-10

194.7 ± 6.7

212.2 ± 6.8

296.9 ± 4.5

101.1 ± 5.9

94.4 ± 2.3

120.2 ± 2.8

158.1 ± 4.8

33.5 ± 3.6 22.8 ± 2.1 26.8 ± 8.2 43.7 ± 4.7 50.3 ± 3.4 62.9 ± 3.5 79.01 ± 3.5 0.424 ± 0.01 0.362 ± 0.07 0.947 ± 0.08 0.386 ± 0.09 0.355 ± 0.04 0.332 ± 0.02 55.26 ± 1.84 59.1 ± 2.14 50.30 ± 2.04 65.12 ± 1.05 66.38 ± 1.2 69.85 ± 3.2

0.424 ± 0.05 67.16 ± 1.04

All values are mean ± SD (n ¼ 3).

space in the lipid bilayer. Hence, the main optimization parameters for preparation of liposomes were the molar ratio of these lipids and the surfactant concentration. Overall, the amount of ANS encapsulated within liposomes depends solely on the type and amount of lipids and surfactants present. Among the conventional and PEGylated liposomes, AL-07 and AL-09 showed maximum EEs followed by the other formulations. Particle size analysis The mean vesicle size and size distribution are the essential parameters that describe the quality of liposome dispersion. The particle size and PDI were determined by using DLS technique and are shown in Table 3. All the formulations, AL-01 to AL-10 showed mean particle sizes ranging from 94.4 ± 4.8 to 296.9 ± 4.5 nm with PDIs ranging between 0.332 ± 0.02 and 0.947 ± 0.08. Formulations AL-01 to AL-03 with increased SPC concentrations showed mean particle sizes of 164.44 ± 4.9 to 183.0 ± 7.1 nm with PDI of 0.671 ± 0.06 to 0.429 ± 0.03, respectively, whereas formulations AL-04 to AL-06 which increased in CH concentrations showed mean particle sizes of 194.7 ± 6.7 to 296.9 ± 4.5 nm with PDIs of 0.424 ± 0.01 to 0.947 ± 0.08, respectively. Finally, formulation AL-07 with 3% v/v Tween-80 showed

the highest EE and had mean particle size of 101.1 ± 5.9 nm with a PDI of 0.386 ± 0.09. Formulations AL-08, AL-09 and AL-10 with increased in MPEG-DSPE2000 concentrations showed mean particle sizes of 94.4 ± 2.3, 120.2 ± 2.8 and 158.1 ± 4.8 nm with PDIs of 0.355 ± 0.04, 0.332 ± 0.08 and 0.424 ± 0.05, respectively. The results indicate that as the lipid (SPC or CH or MPEG-DSPE2000) concentration increased, the particle size also increased. Addition of 3% (v/v) Tween-80 for the AL-07 to AL-10 formulations resulted in the decrease of particle size when compared to all other formulations. There was no significant change in liposome particle size and PDI before and after freeze drying, suggesting that the freeze drying cycle used was optimum and the formulations contained sufficient amount of lyoprotectant to preserve the integrity of the liposomes. A typical phenomenon of instability in the liposome formulation is the increase in particle size due to the aggregation or the fusion of unstable liposomes during the formulation processing and/or upon storage. An increase in particle size of liposomes generally results in rapid uptake by the reticuloendothelial system (RES) with subsequent rapid clearance and a short half-life (Gu et al., 1982; Jain et al., 2003). Thus, controlling and maintaining liposomes at small and uniform size are vital in developing a possible pharmaceutical product. It was interesting to observe that the particle size of liposomes

8

G. V. Shavi et al.


before and after freeze drying were almost constant when 3% (v/v) Tween-80 was added in the hydration medium. These results suggested that the incorporation of Tween-80 resulted in the increase of the liposome stability in the solution. This may be due to the hydrocarbon tail of Tween-80 being able to penetrate into the lipid bilayer, thus leaving the polyethylene oxide groups on the surface of the liposomes thereby introducing a steric barrier on the surface of the liposomes, which might decrease liposome fusion and consequently decrease lipid and ANS exchange upon collision of the liposome particles (Chen et al., 2007; Yang et al., 2007a,b,c). Overall, the results suggested that the particle sizes of AL-07 and AL-09 were suitable for intravenous administration. Zeta potential The stability of liposomes was assessed by measuring the zeta potential of the particles by Zetasizer Nano ZS. If all the particles in suspension have a large positive or negative zeta potential, then they will tend to repel each other and there will be no tendency for the particles to come together. Zeta potential measurements showed that the differences in vesicle size might exert an influence on the charges carried by the vesicles. The results of zeta potential for different formulations are shown in Table 3. All the formulations from AL-01 to AL-10 showed zeta potential value ranging from 22.8 ± 2.1 to 79.01 ± 3.5 mV. Formulations AL-01 to AL-03 with increased SPC concentration showed zeta potential values of 26.46 ± 2.9 to 32.75 ± 6.7 mV and formulations AL-04 to AL-06 with increased CH concentrations showed zeta potential values of 33.5 ± 3.6 to 26.8 ± 8.2 mV, respectively. Thereafter, for formulation AL-07 having 3% v/v Tween-80 with a mean particle size of 101.1 ± 5.9 nm and a PDI of 0.386 ± 0.09 showed a zeta potential value of 43.7 ± 4.7 mV and formulations AL-08, AL-09 and AL-10 with an increased MPEG-DSPE2000 concentrations showed 50.3 ± 3.4, 62.9 ± 3.5 and 79.01 ± 3.5 mV zeta potential values, respectively. The results of zeta potential for formulations AL-07 and AL-09 having maximum EE are depicted in Figure 1. The results indicate that the increase in SPC or MPEG-DSPE2000 concentration and addition of 3% v/v Tween-80 resulted in more negative zeta potential values. Although Tween-80 is a non-ionic surfactant, it can be partially hydrolyzed in acidic or basic conditions, making the liposome surface negatively charged. Moreover, the negative charge on the liposome surface also can decrease the attraction among the liposome particles by offering repulsive forces and thereby stabilize the liposome by preventing their aggregation. Increases in the CH concentrations showed lower negative zeta potential values. There was no significant difference in zeta potential values before and after freeze drying. The zeta potential of PEGylated liposome was more negative than that of conventional liposomes due to the negatively charged phosphate group of MPEG-DSPE2000, which is also in accordance with results reported in the literature (Hinrichs et al., 2006; Yang et al., 2007a,b,c). In this case, the effect of Tween-80 on the zeta potential seems to be negligible since the negative charge due to the PEGylation is so much larger and also PEGylated lipid


might be liable to distribute into the outer monolayer, leading to a more negative charge. Overall, the results suggested that these liposomes are stable and did not aggregate upon storage for longer period, i.e. at 5 ± 3 C of storage for 6 months. FTIR spectroscopy The FTIR and DSC analysis was executed to assess state of drug when encapsulated into liposomes. In the present study, it majorly depicts the crystalline/amorphous (DSC) and chemical changes (FTIR) of the molecule those ascend from the formulated lipids carrier system. The FTIR results of pure drug, blank and drug-loaded liposomes (AL-07 and AL-09) containing major absorption band peaks are depicted in Figures 2 and 3. FTIR spectra of the drug show major characteristic absorption bands peaks at 3099.7 cm1 due to aromatic C–H stretch, 2982.05 and 2918.4 cm1due to CH3 aliphatic stretch, 2234.64 cm1 (medium CN stretch), 1604.83 cm1 (skeletal vibrations) due to C ¼ C of aromatic ring, 1572.04 and 1502.6 cm1 due to aromatic ring, 1537.32 cm1 due to C ¼ N stretching, 1465.95, 1357.93 and 1390.72 cm1 due to gem dimethyl groups, 1273.06 and 1201.69 cm1 due to tertiary butyl type skeletal vibration, and 761.91 cm1 due to out of plane C–H band (aromatic ring), respectively. The FTIR spectra of the blank liposomes did not show major peaks in the regions of the drug spectrum. In the blank AL-07 and AL-09 liposomes, peaks obtained in the spectra with less intensity were due to interactions of lipids. The wave numbers obtained for AL-07 liposomes are 3419.90, 2924.18, 2357.09, 2332.02, 2135.27, 1735.99, 1633.76, 1238.34, 1084.03 and 719.47 cm1. Similarly the wave numbers obtained for AL-09 are 3462.34, 3423.76, 2922.25, 2353.23, 2322.37, 2065.83, 1734.06, 1633.76, 1454.38, 1367.58, 1238.4, 1082.10 and 721.40 cm1. The major peaks with maximum intensities of drug shifted to lower frequencies with lower maximum intensity (energy required for stretching becoming less) resulting from broadening of peaks was observed in the AL-07 and AL-09 liposomes which indicates an interaction between the drug and the lipids. Thus, the results of FTIR spectroscopy support the formation of a dispersed phase of drug molecules in the lipid bilayer structures which is the characteristics of freshly prepared freeze dried AL-07 and AL-09 liposomes. Further, the results of this study were also supported by DSC studies described in the next section. DSC analysis In this study, DSC was used to confirm that the presence of ANS stabilized in the lamellar structure of the bilayer by increasing the transition temperature, which is a useful tool in the investigation of physical state interactions between the drug and the lipids. DSC experiments involved the study of thermal behavior for pure drug, blank and drug-loaded (AL-07 and AL-09) liposomes. The results of the same with melting peaks and heat of enthalpy compared to pure drug are shown in Figures 4 and 5. The DSC thermogram of ANS exhibited a sharp endothermic peak at 83.87 C, corresponding to its melting peak with a DH ¼ 131.12 mJ. The peak corresponding to the drug did not appear in any thermogram of prepared blank (AL-07 and AL-09) liposomes. The melting


9


DOI: 10.3109/08982104.2015.1029493

Figure 2. FTIR spectra of (a) ANS, (b) conventional blank and (c) AL-07 liposomes.

peaks obtained for drug-loaded (AL-07 and AL-09) liposomes were 120.88 and 103.92 mJ with a DH values of 25.2 and 65.98 mJ, respectively, with low intensity and broad endothermic peaks compared to the pure drug. This indicates the reduced crystallinity of drug in the lipid bilayer structures forming a new phase. The results of DSC analysis for drugloaded AL-07 and AL-09 liposomes indicate that the intensity of endothermic peaks observed were signifying that all the lipid components interact with each other to a great extent while forming the lipid bilayer, diminished and appearance of distinct peaks, with a different melting transition from that

observed with either of the individual components, resulting in formation of a new phase. The incorporated ANS associated with lipid bilayers has interacted to a large extent with them. Absence of the melting endotherm of ANS and shifting of the lipid bilayer components endotherm suggested significant interaction of ANS with the bilayer lipid. The shift of endothermic peak in the liposomes suggests that a thermodynamically stabilizing interaction of all components leading to enhanced encapsulation of the drug and a decreased rate of release. Thus, the results clearly show that the melting peak of the drug can be shifted through formation

G. V. Shavi et al.


10


Figure 3. FTIR spectra of (a) ANS, (b) PEGylated blank and (c) AL-09 liposomes.

Figure 4. DSC curves of (a) ANS, (b) conventional blank and (c) AL-07 liposomes.

of liposomes. The thermograms of any type of the prepared liposomes containing drug indicate that drug existed in a new (amorphous) phase (Hashizaki et al., 2003; Hathout et al., 2007; Momo et al., 2005; Nicholov et al., 1995) in the lipid bilayer structures and it would be evenly dispersed at the molecular level in the freshly prepared freeze dried liposomes. NSTEM analysis The surface morphology of the prepared liposomes of formulation, AL-07 and AL-09 are shown in Figure 6(a) and (b). The prepared liposomes were well-identified with a

Figure 5. DSC curves of (a) ANS, (b) PEGylated blank and (c) AL-09 liposomes.

spherical diameter. TEM analysis demonstrated that conventional and PEGylated liposomes retained then hydrated morphology as near spherical morphology and a discrete nature was observed for both. A vesicular structure was discernable, whereas the inner lamellar could not be unambiguously observed (Chen et al., 2009; Dubey et al., 2007; Nounou et al., 2006). Comparison of the photographs indicated no obvious changes in both liposome morphologies, as a result of the drug encapsulation. The image from negative staining TEM confirmed that liposomes were round, smooth, free from drug crystals, multilamellar of similar morphology in nature for all formulations and the lamellae were clearly


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DOI: 10.3109/08982104.2015.1029493

Figure 6. (a) TEM analysis of conventional liposomes (AL-07) at 120 000. (b) TEM analysis of PEGylated liposomes (AL-09) at 120 000.

visible. Therefore, it seems that ANS encapsulation did not significantly affect the morphology of the liposomes. It also revealed that presence of homogenous population of multilamellar vesicles that consist of many concentric phospholipid bilayers. In vitro release study ANS release from liposomes changed with type and concentration of lipids used in their preparations. The in vitro release

studies of AL-01 to AL-04 and AL-07 to AL-10 formulations were studied and the results of the same were depicted in Figures 7 and 8. The formulations AL-01 to AL-04 showed 51.98 ± 1.48 % to 96.23 ± 2.5% drug release after 24 h. Thereafter, formulations AL-07, AL-08, AL-09 and AL-10 showed drug release of 46.01 ± 4.8%, 41.23 ± 1.9%, 24.01 ± 3.8% and 23.54 ± 1.2% after 24 h, respectively. A rapid release was obtained for AL-01 and AL-02 formulations with low lipid concentration followed by AL-03 formulation. There was not much of a significant difference in drug release


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G. V. Shavi et al.

Figure 7. In vitro drug release profile of AL-01, AL-02, AL-03 and AL04 conventional liposomes.


the encapsulated drug substance. In addition, it takes time for ANS to be released once encapsulated in the liposomes because lipid bilayers are stabilized by CH and/or Tween-80 (Deol & Khuller, 1997; Srinath et al., 2000; Yang et al., 2009). Thus, a depot effect could be achieved using liposomes, especially in the PEGylated liposomal formulation. In vitro drug release of the conventional liposomes followed the order AL-014AL-024AL-034AL-04 and AL-07 and for the PEGylated liposomes order of drug release was found to be AL-084AL-094AL-10. So, the overall results suggest that the lipids used in the study exhibited a biphasic release behavior. The liposomes would be stable in the blood circulation for longer periods and the drug would be released slowly at the tumor site. These results indicate that the PEGylated (AL-09) liposomal formulations meet the requirement for a sustained drug delivery system compared to the conventional (AL-07) liposomal formulations. Overall R2 values for both AL-07 and AL-09 formulations can be attributed to Higuchi model release pattern. Korsemeyer– Peppas n-value 0.55n51.0 suggests the anomalous (nonFickian) transport. This can be further depicted as outward diffusion of the drug molecules from the lipid embedded matrix. The relevant processes are penetration of dissolution medium into the lipid matrix, lipid hydration and simultaneous drug release. Variation in lipid concentrations may modify the drug release (Arica et al., 1995; Bodde´ & Joosten, 2006; Dodov et al., 2004). In vitro cytotoxicity studies in human breast cancer cells

Figure 8. In vitro drug release profile of AL-07 (conventional liposomes), AL-08, AL-09 and AL-10 (PEGylated liposomes).

for the AL-04 and AL-07 formulations for the liposomes AL-08, AL-09 and AL-10 prepared with addition of MPEG-DSPE2000 in percentage drug release was reduced (controlling the release rate). This can be attributed to the slower diffusion of drug from liposomes. There was a sharp increase in the drug release after 24 and 72 h for conventional liposomes (AL-01 to AL-04 and AL-07) and PEGylated liposomes (AL-08 to AL-10), respectively. The more rapid drug release is due to leakage of drug from the lipid bilayer, which acts as a rate-limiting membrane for release of the encapsulated drug. For the PEGylated liposomes, AL-09 showed only 65.13 ± 2.3% drug release when compared to AL-07 which showed 92.78 ± 3.7 drug releases after 72 h. After 168 h almost complete drug release (490%) was observed for all formulations, excluding AL-09 and AL-10. From the results, conventional and PEGylated liposomes (AL-01, AL-02, AL-03, AL-07 and AL-08 to AL-10) showed an initial fast release followed by sustained release (diffusion processes), indicating that the release of ANS reached a slow release status (Kim et al., 2006). The rate of ANS release reduced with the increasing concentration of lipids (SPC or MPEG-DSPE2000) due to a high an encapsulation of drug in the lipid bilayers structures. This behavior could be related to the decreased permeability of the phospholipids bilayer for

Anticancer effects of ANS-loaded liposomes, AL-07 and AL09 (optimized formulations) compared to pure drug were evaluated in human breast cancer cells, especially in ERpositive and tumorigenic breast cancer cell lines like MCF-7 and BT-549. A methyl tetrazolium (MTT) assay (Sargent, 2003) was performed by incubation at various time points (48, 72 and 168 h) and IC50 values for pure drug and liposomal formulations were compared. The IC50 values were determined from concentration versus percentage cell viability plot. In all the experiments, pure drug and optimized formulations (AL-07 and AL-09) were suspended in PBS sterilized by passing through a 0.22 mm syringe filter. Cells without any treatment were used as negative control (Immordino et al., 2003). The results of the IC50 values are expressed in mean ± SD (n ¼ 3) for optimized liposomal formulations in comparison with pure drug are shown in Figures 9(a)–(c) and 10(a)–(c). A concentration dependent decline of the percentage cell viability was observed upon treatment with the pure drug and liposomes (AL-07 and AL-09) at all the studied time points. The IC50 value of pure drug against MCF-7 cells was 1.2, 1.5 and 7.0 mg/mL and similarly, in BT-549 cells IC50 value found to be 0.26, 0.35 and 3.0 mg/mL at 48, 72 and 168 h of incubation, respectively. Both liposomes (AL-07 and AL-09) were found to have comparable cytotoxic effects in MCF-7 and BT-549 cells at various time points tested. In MCF-7 cells IC50 of AL-07 were found to be 410, 2.0 and 0.49 mg/mL at 48, 72 and 168 h, respectively. Similarly, for AL-09, IC50 were found to be 3.0, 1.0 and 0.31 mg/mL, respectively. In BT-549


Figure 9. (a) Percentage cell viability of pure drug, AL-07 and AL-09 liposomes at 48 h in BT-549 cell lines. (b) Percentage cell viability of pure drug, AL-07 and AL-09 liposomes at 72 h in BT-549 cell lines. (c) Percentage cell viability of pure drug, AL-07 and AL-09 liposomes at 168 h in BT-549 cell lines. p50.05, significantly compared to pure drug and results are expressed in mean ± SD (n ¼ 3).


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Figure 10. (a) Percentage cell viability of pure drug, AL-07 and AL-09 liposomes at 48 h in MCF-7 cell lines. (b) Percentage cell viability of pure drug, AL-07 and AL-09 liposomes at 72 h in MCF-7 cell lines. (c) Percentage cell viability of pure drug, AL-07 and AL-09 liposomes at 168 h in MCF-7 cell lines. p50.05, significantly compared to pure drug and results are expressed in mean±SD (n ¼ 3).


14 J Liposome Res, Early Online: 1–19


DOI: 10.3109/08982104.2015.1029493

cells, IC50 of AL-07 was found to be 10, 3 and 0.42 mg/mL at 48, 72 and 168 h. Similarly, for AL-09, it was 1.7, 1.8 and 0.8 mg/mL, respectively. ANS as free and encapsulated forms (liposomes) exhibited better cytotoxicity against BT-549 cell lines compared to MCF-7 cell lines at all the exposure time points. From these results, it is evident that cytotoxic effect of pure drug at 168 h treatment is diminished which may due to depletion of drug in the culture medium. However, for the AL-07 and AL-09 liposomes, the 48 and 72 h assays showed considerable changes in their cytotoxicity activities. The present study suggests that a concentration-dependent reduction in the cell viability for both the pure drug and liposomal formulations. Pure drug was found to have similar IC50s for both the 48 and 72 h assays, however, 168 h, IC50 values were increased. The cytotoxic activity of the drug may reduce at longer incubation time due to shorter half-life (Sarkar & Yang, 2008) of the drug. The surviving fraction of cell the population is able to proliferate in the cell culture at longer incubation times and in turn increases the percentage of viable cells. Liposome (AL-07 and AL-09) formulations showed a reduction in cell viability for the 48 or 72 h assays when compared with the pure drug. However, it is striking to note that at the longer incubation time (168 h), the cytotoxic effect significantly increased and percentage of viable cells remained less as compared to the pure drug. This remarkable enhancement in cytotoxicity is due to the slow release of the drug from the lipid bilayer. When compared to AL-07, the PEGylated liposomes (AL-09) showed more cytotoxic at 168 h and the number of viable cells was found to be less at a concentration of 10 mg/mL (p50.05). The anticancer effects of AL-09 remain constant for the longer incubation time tested (168 h), but in the case of conventional liposomes, AL-07 a decline in the activity would be expected after 168 h of incubation. These data suggest that a prolonged anticancer effect of PEGylated liposomes when compared with conventional liposomes (Crosasso et al., 2000; Turanek et al., 2009). The available dose of the drug could


15

be reduced when formulated in a liposome system for the better delivery with a minimum or reduced adverse effect induced by the anticancer drug, ANS. This elevated cytotoxicity of PEGyalted liposomes at longer incubation time points may be due to the sustained and slower release of drug from the liposomes. Similar results were observed for PEGylated liposomes when tested against MCF-7 cell lines (Liu et al., 2008). Pharmacokinetic studies of optimized formulations In order to study the in vivo performance, the pure drug and developed formulations liposomes were administered to six rats separately. The plasma concentration time profiles after single intravenous administration of pure drug and ANSloaded liposomes AL-07 and AL-09 at a dose of 1 mg/kg in rats are depicted in Figure 11. The results are expressed in mean ± SD of pharmacokinetic parameters, AUC(0!1), clearance, volume of distribution, MRT and t1/2 are summarized in Table 4. Pharmacokinetic profiles of pure ANS is characterized by AUC(0–1) value of 21.0 ± 1.34 mg/h/mL, MRT value of 12.6 ± 1.8 h and clearance value of 0.0475 ± 0.014 L/h/kg. There was a significant difference (p50.001) in the pharmacokinetic parameters when ANS was formulated in the form of liposomes. PEGylated liposomes had comparatively better pharmacokinetic profiles than conventional liposomes. Both liposomal formulations had displayed (2-fold) reduction in the initial time points than pure drug indicating sustained drug delivery of ANS from liposomes. This would avoid higher initial concentration in the systemic circulation thereby reduce the systemic related toxicity effects. Conventional and PEGylated liposomes demonstrated a 3.33- and 20.28-fold increase in AUC(0–1) values, respectively, when compared to pure drug. At the same time, ANS-loaded conventional and PEGylated liposomes have shown 7- and 10-fold lower elimination rate than pure drug. Lower elimination rate is probably due to sustained release of drug from the encapsulated vesicles which was further

Figure 11. Plasma drug concentration versus time profile of pure drug, AL-07 and AL-09 liposomes (y-axis – represented in log scale). p50.001, significantly compared to pure drug and conventional liposomes and expressed in mean ± SD (n ¼ 6).

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Table 4. Pharmacokinetic parameters of pure drug, AL-07 and AL-09 liposomes (n ¼ 6). Mean ± SD Parameters

Pure drug (intravenous route)

AL-07 (conventional liposomes)

AL-09 (PEGylated liposomes)

8.3 ± 0.98 21.0 ± 1.34 0.0763 ± 0.012 0.0475 ± 0.014 0.623 ± 0.02 12.6 ± 1.8 9.08 ± 1.46

4.2 ± 0.86* 70.1 ± 2.14* 0.011 ± 0.0013* 0.014 ± 0.0011* 1.242 ± 0.04* 63.3 ± 2.75* 60.4 ± 4.85*

4.3 ± 0.89* 426.4 ± 3.12* 0.007 ± 0.0015* 0.002 ± 0.0003* 0.323 ± 0.07* 140.4 ± 5.3* 95.60 ± 5.84*

Cinitial (mg/mL) AUC(0–1) (mg h/mL) Kel (h1) Cl (L/h/kg) Vd (L/kg) MRT (h) t1/2

*p Value50.001 at 99% confidence interval.


Table 5. Percentage EE, mean particle size, PDI and zeta potential of AL-07 and AL-09 formulations at 25 C/60 ± 5% RH.

Time (months)

% EE

AL-07 formulation Initial 65.12 ± 1.05 0.5 64.34 ± 1.56 1 63.02 ± 1.43 2 60.76 ± 1.59 3 57.56 ± 5.5 6 52.12 ± 1.4 AL-09 formulation Initial 69.85 ± 1.9 0.5 68.89 ± 1.4 1 67.73 ± 1.3 2 65.3 ± 1.6 3 61.8.4 ± 1.2 6 55.18 ± 1.9

Particle size (nm)

PDI

Zeta potential (mV)

101.1 ± 5.9 118.7 ± 7.6 139.5 ± 4.5 176.4 ± 2.6 199 ± 6.5 260 ± 5.76

0.386 ± 0.09 43.7 ± 4.7 0.395 ± 0.03 40.1 ± 3.2 0.407 ± 0.06 37.65 ± 6.5 0.419 ± 0.05 29.98 ± 4.4 0.428 ± 0.03 24.6 ± 3.8 0.456 ± 0.08 17.3 ± 6.67

120.2 ± 2.8 133.8 ± 6.7 148.8 ± 3.7 205.7 ± 4.5 248.6 ± 5.4 310 ± 4.6

0.332 ± 0.02 62.9 ± 3.5 0.345 ± 0.04 57.6 ± 5.5 0.361 ± 0.06 51.8 ± 3.2 0.384 ± 0.07 48.61 ± 5.2 0.422 ± 0.01 37.3 ± 3.7 0.494 ± 0.05 21.2 ± 2.63

All values are mean ± SD (n ¼ 3). Degradation constant (k) for AL-07 ¼ 0.0161, degradation constant (k) for AL-09 ¼ 0.0184.

supported by the in vitro release profile. Clearance values of ANS were greatly altered with 3.39- and 23.75-fold reduction in the clearance rate than the pure drug (Drummond et al., 1999; Guo et al., 2005; Yang et al., 2007a,b,c). PEGylated liposomes showed smaller volumes of distribution and conventional showed slightly larger volumes of distribution as compared to that of pure drug. Liposomal formulations also revealed significantly higher mean residence times (5.02and 11.11-fold) and longer biological half-lives (6.71- and 10.62-fold) for conventional and PEGylated liposomes, respectively, compared to pure drug. The present study results were in agreement with the earlier reports (Fetterly & Straubinger, 2003; Junping et al., 2000; Yang et al., 2007a,b,c). It has been reported that PEGylated liposomes could provide sustained release, longer blood circulation halflives, higher systemic exposures, lower clearance rates and smaller volumes of distribution. The reason for the longer blood circulation half-lives is due to hydrophilic nature of PEGylated liposomes which results in reduced interactions with opsonin/blood proteins in vivo and thus reduced RES uptake leading to prolonged circulation (Zhao et al., 2009). Briefly, PEGylated liposomal nanocarriers have ability to evade the RES and extend the circulation time of encapsulated drugs in the bloodstream. PEG chains on the outer leaflet of the liposomal bilayer are thought to provide a steric barrier to opsonin binding resulting in RES evasion. In such type of systems, prolonged circulation in the bloodstream results in enhanced extravasation at sites exhibiting increased

vasculature permeability. So, liposomes/PEGylated liposomes may mostly accumulate into cancerous cells avoiding healthy cells. Unlike, the free drug or conventional formulations may not only go into cancerous cells and tissues of the body but may also get exposed to healthy cells and tissues causing undesired adverse events and tissue damage. Also, in many research studies it is depicted same, that liposomes when formulated with inclusion of PEG’s, due to their nanometer particle size range get localized and accumulated in tumors via ‘‘leaky’’ vasculature through the enhanced permeability and retention effect. This well-known phenomenon results in accumulation of liposomal nanocarriers at sites with compromised vasculature. Results of ANS-loaded PEGylated liposomes exhibited better pharmacokinetic profiles than conventional liposomes and the pure drug. PEGylated liposomes are found to be an ideal sustained delivery system for ANS which could reduce the dosing frequency, maintaining the plasma levels for longer time and avoidance of toxicity. It could be concluded that ANS-loaded PEGylated liposomes might be an alternative sustained drug delivery system for passive targeting into the tumor site. Switching to a liposomal formulation offers the potential to alter the pharmacokinetics of ANS and provide a more favorable efficacy for this promising chemotherapeutic agent. Stability studies The stability is one of the major obstacles for the formulation of liposomes because of aggregation and leaching of drug from lipid layers. Moreover liposomes are made up of lipids since it is easy to oxidize; hence the stability is the major problem for liposomes storage/shipment (Bhalerao & Harshal, 2003; Carstensen, 1995). All the analyses were carried out in triplicates and the results are reported as mean ± SD. The results of physical stability of AL-07 and AL-09 for various lengths of time are shown in Table 5. On storage as per ICH guidelines at the accelerated stability condition, 25 ± 2 C/ 60 ± 5% RH, for AL-07 and AL-09, liposomes showed slight change in physical appearance (leaching of drug from lipid bilayer and slight aggregation), significant change in percentage encapsulation, which was decreased more than 5% of original value observed by HPLC analysis after storage period of 2 months and decreased more than 10% of original after storage period of 6 months study, respectively. The particle size distributions, PDIs and zeta potentials for these formulations at accelerated storage condition were quite high, but were stable for 60 short days. After that both formulations



DOI: 10.3109/08982104.2015.1029493

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Figure 12. log % drug remaining versus time plot of AL-07 and AL-09 liposomes at 25 C/60%RH.

were less stable in a study period of 6 months. The graph was plotted by using log % drug remaining versus time. The results of the same are depicted in Figure 12. Shelf-life calculated by linear extrapolation by first-order plot was found to be 6 months 16 days and 5 months 21 days for AL-07 and AL-09 (exhibited similar stability) with degradation constant (k) value of 0.0161 and 0.084, respectively (Deshpande et al., 2010). The optimized storage condition for formulations, AL-07 and AL-09, was found to be stable at 5 ± 3 C. The proposed shelf-life (6 months at 5 ± 3 C) is based on available laboratory conditions at small scale. However, the stability conditions and shelf-life may positively be improved by adopting superior and large scale production oriented techniques, related to ease of manufacturing process.

Conclusion In this study liposomes, both conventional and long circulating (PEGylated liposomes), prepared thin film hydration method using different molar ratios of drug to lipids (SPC, CH and MPEG-DSPE2000). PEGylated liposomes (AL-09) shows higher drug EEs with desired particle size distribution, polydispersities with enhanced cytotoxic effects at the end of the 168-h incubation time against breast cancer BT549 and MCF-7 cell lines as evidenced by decreases in the IC50values with higher stability at 5 ± 3 C in comparison to the free and conventional liposomes of ANS. The pharmacokinetic studies demonstrated higher values of AUC(0–1) with increased plasma half-life for PEGylated liposomes indicating their ability to be retained in the circulation for prolonged periods of time evading RES uptake. For that reason, PEGylated liposome formulation of ANS could provide a good alternative and suitable carrier for large scale production with sustained delivery properties for effective treatment of breast cancer.

Acknowledgements The authors would like to acknowledge Sun Pharma, Baroda, India, for gift sample anastrozole and the All India Council for Technical Education (AICTE), Govt. of India, for providing National Doctoral Fellowship for this project.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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