Cellular uptake, cytotoxicity and in-vivo evaluation of Tamoxifen citrate loaded niosomes

Accepted Manuscript Title: Cellular uptake, cytotoxicity and in-vivo evaluation of Tamoxifen citrate loaded niosomes Author: Dalia S. Shaker Mohamed A. Shaker Mahmoud S. Hanafy PII: DOI: Reference:

S0378-5173(15)30060-0 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.07.041 IJP 15045

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

28-5-2015 14-7-2015 15-7-2015

Please cite this article as: Shaker, Dalia S., Shaker, Mohamed A., Hanafy, Mahmoud S., Cellular uptake, cytotoxicity and in-vivo evaluation of Tamoxifen citrate loaded niosomes.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.07.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cellular uptake, cytotoxicity and in-vivo evaluation of Tamoxifen citrate loaded niosomes Dalia S. Shakera,b*, Mohamed A. Shakera, Mahmoud S. Hanafya. a Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Cairo, Egypt. b Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences and Pharmaceutical industries, FUE, Cairo, Egypt.

* Corresponding to: Dalia Samuel Shaker, PhD. Future University, Pharmaceutical Technology Department, College of Pharmacy, Cairo, Egypt Tel: (202) - 260-67419 Fax: (202) -2618-6111 P.O. Box: 11477 Email: [email protected], [email protected] Emails of Co-authors - Mohamed A. Shaker: [email protected] - Mahmoud S. Hanafy: [email protected]

abstract Graphical

Abstract

One of the main challenges in Tamoxifen cancer therapy is achieving localized, efficient and sustained delivery without harming normal healthy organs. This study focused on evaluating Tamoxifen Citrate (TMC) niosomes for localized cancer therapy through in-vitro breast cancer cytotoxicity as well as in-vivo solid anti-tumor efficacy. Different niosomal formulae were prepared by film hydration technique and characterized for entrapment efficiency % (E. E), vesicle size, morphology, and invitro release. The cellular uptake and anti-cancer activity were also tested in-vitro using MCF-7 breast cancer cell line. Moreover, in-vivo anti-tumor efficacy was examined in Ehrlich carcinoma mice model through reporting solid tumor volume regression and tissue TMC distribution. The obtained niosomes prepared with Span 60: cholesterol (1: 1 molar ratio) showed a distinct nano-spherical shape with EE up to 92.3% ± 2.3. Remarkably prolonged release of TMC following diffusion release behavior was detected. The optimized formula showed significantly enhanced cellular uptake (2.8 fold) and exhibited significantly greater cytotoxic activity with MCF-7 breast cancer cell line. In-vivo experiment showed enhanced tumor volume reduction of niosomal TMC when compared to free TMC. Based on these results, the prepared niosomes demonstrated to be promising as a nano-size delivery vehicle for localized and sustained TMC cancer therapy.

Keywords Tamoxifen - Cancer - Niosomes - MCF-7 - Cellular uptake - Cytotoxicity

Chemical compounds stated in this article Tamoxifen Citrate (PubChem CID: 2733525); Span 60 (PubChem CID: 14455022); Span 40 (PubChem CID: 11502266); Span 80 (PubChem CID: 54085240); Tween 20 (PubChem CID: 443314); Cholesterol (PubChem CID: 5997).

1. Introduction

Over the last decade as the number of people who diagnosed with cancer increased, with the understanding for the role of hormones in cancer growth, extensive research studies have illustrated the effectiveness of hormone antagonists as cancer therapy (Ferlay, 2013; Murray, 2010; Ban and Godellas, 2014). Tamoxifen citrate (TMC) as one of these hormone antagonists, is commonly indicated to treat breast cancers that are estrogen receptor positive (ER+) (Cole et al., 1971). Tamoxifen Citrate is a non-steroidal anti-estrogen that modulates the intracellular estrogen receptors present in the breast tissue, so it stops cell proliferation and growth. Hence, it is currently prescribed in treating early, advanced (metastatic) breast cancer in both pre- and post- menopausal women, as well as, a hormone therapy to treat male breast cancer (Dutertre and Smith, 2000; Howell et al., 2000). Although, the effectiveness of TMC therapy has been extensively investigated and approved, it still possesses many problems that decrease patient compliance or adherence to the therapy and restrict its full clinical relevance. Oral TMC therapy has always been associated with poor bioavailability due to poor aqueous solubility and its liability to intestinal metabolism as well as rapid clearance with the first pass metabolism. Moreover, prolonged systemic TMC therapy is associated with life-threatening oxidative stress mediated hepatic toxicities, ototoxicity (Pillai and Siegel, 2011), thrombo-embolic events, cataract, retinopathy and reduced cognition, as well as increases the incidence of endometrial cancer and liver cancer (Yang et al., 2013). In spite of the aforementioned challenges, an extensive research had been focused on the development of different TMC delivery strategies trying to fulfill two main purposes. The first is to maximize its therapeutic efficacy and minimizing its unwanted side effects post-administration to the body. The second is the sustained and localized delivery of TMC to its site of action, to improve drug pharmacokinetics and the way drug is administered. Such delivery systems include but not restricted to the use of liposomes (Cosco et al., 2012), micelles (Licciardi et al., 2010), dendrimers (Li et al., 2012), and polymeric microspheres (Coppi and Lannuccelli, 2009; Kivadiya et al., 2012)/ nanospheres (Jain et al., 2011) as injectable controlled release systems, as well as, ethosomes for transdermal delivery (Sarwa et al., 2013). On the other hand, niosomes as a brilliant pharmaceutical carrier employed as a drug-encapsulating vehicles to target deliver anticancer drugs in a controlled and /or sustained manner for definite periods, ranging from hours to weeks (Sezgin-Bayindir

et al., 2013; Tavano et al., 2013).They can easily be obtained by the self-assembly of non-ionic surfactants in bi-layered structure to form vesicles with or without cholesterol as an additive (Arunothayanun et al., 2000; Bragagni et al., 2014). Such a quickly formulated bilayer carrier provides advantages over the other delivery vehicle. Mainly, sufficient stability to these self-assembled micro/nano-sized, biodegradable, biocompatible and non-immunogenic vesicles against collapse during application. Keeping their well-defined configuration in space is crucial and specifically needed for the delivery of drug(s) that should be secured or protected from the uncontrolled burst release during their in-vivo circulation. Niosomes not only slow down the release of the loaded drugs but also tailor the release rate and pattern through adjusting the bilayer composition and cholesterol content. In addition to their high encapsulation efficiency for both hydrophilic (aqueous core) or lipophilic (bilayer membrane) drug, they enhance localization of the drug into target site by enhancing cellular uptake passively or actively with minimal side effects (Bendas et al., 2013; Moghassemi and Hadjizadeh, 2014). To the best of our knowledge, no research study has been done on niosomes for the localized and sustained delivery of TMC and is examined in this study for the first time. Herein, the evaluation of niosomal TMC performance in comparison to free TMC regarding in-vitro cellular uptake and cytotoxicity using MCF-7 breast cancer cell line. In-vivo behavior through induction of solid tumor, measurement of tumor volume and TMC distribution in different organs was assessed as well.

2. Material and methods 2.1 Material Tamoxifen Citrate (purity 98.9%) was kindly gifted by Medical Union Pharmaceuticals; Egypt. Span 60 was purchased from Sigma Aldrich, USA. Span 40 and Span 80 were obtained from Oxford laboratories, India, whereas Tween 20 and cholesterol were obtained from Winlab, UK. HPLC grade methanol and acetonitrile were purchased from Sigma Aldrich, Germany, whereas chloroform was purchased from United Company for medical and chemical preparations, Egypt. MCF-7 cell line was kindly obtained from the National Cancer Institute (NCI), Egypt. RPMI1640 culture media was purchased from Lonza Company for biological products, USA.

Other chemicals used were reagent grade and all of the chemicals were used as received without any further purification. 2.2 Preparation of Tamoxifen citrate loaded niosomes Niosomal formulae were prepared by thin film hydration method as described before (Guinedi et al., 2005). Using 250 ml round flask, 30 mg of TMC and 300 mg of

non-ionic

surfactant

(NIS)

and

cholesterol

(CH)

were

dissolved

in

chloroform/methanol mixture (2:1 v/v). The total mixture was then rotary evaporated under reduced pressure at 40°C, until the thin film was formed and then kept overnight under room air and temperature. The dried thin layer was then hydrated with 30 ml of Phosphate buffer (PB) of pH 7.4 at temperature just above the glass transition temperature of each surfactant. It was 45°, 60°, 15° and 2°C for Span 40, Span 60, Span 80 and Tween 20, respectively. The formed niosomal suspension was then sonicated using probe sonicator for 3 min. The prepared drug loaded niosomes was separated from un-entrapped TMC by centrifugation of the niosomal suspension at speed of 20,000 rcf and 4°C for 60 minutes. The collected niosomes were washed three times with PB to separate the un-entrapped from entrapped TMC, re-suspended again in the PB and the prepared niosomal formulations named based on the used NIS and its molar ratio with CH (as illustrated in table 1). 2.3 Determination of entrapment efficiency The entrapment efficiency (E. E) was directly determined by measuring the drug content present in the niosomes obtained after centrifugation and washing. One ml of the niosomal dispersion was re-centrifuged at 20,000 rcf for 30 min. The supernatant was discarded and the niosomal pellets were dissolved and vortexed with 10 ml of pure methanol for 3 min in order to disassemble the niosomal particles and dissolve all the entrapped TMC. The UV absorbance of methanol solution was determined at 276 nm using a UV/visible spectroscopy (Jasco spectrophotometer, Japan) (The British Pharmacopoeia, 2009). The percentage of the E. E was calculated as follows (Hao et al., 2011): E. E% = (amount of encapsulated drug/ initial amount of added drug) × 100 (1) 2.4 In-vitro TMC release kinetics

The release of TMC was performed using the dialysis method as described by (Hao et al., 2002; Paolino et al., 2008). An appropriate volume (3 ml) of niosomal suspensions of conc 1mg/ml was sealed in dialysis bag (MWCO 12000-14000) of 5 cm length and 2.1 cm wide. The dialysis bag was then immersed in 40 ml PB containing 0.1% (w/w) Tween80 (pH 7.4) as a release medium that was 3-5 times greater in respect to the saturation solubility of the drug to ensure un-impaired dissolution to comply to the sink condition (Mahmoudi et al., 2014) (the solubility of TMC in PB containing 0.1% Tween 80 was 0.32 mg/ml). The release medium kept under continuous agitation using digital shaker water bath and maintained at 37°C. At various time intervals and up to 6 days, 1 ml sample was withdrawn out and replaced with 1 ml of fresh medium. The release of free TMC was done at the same condition as a control. Samples were analyzed for UV absorbance at a wave length of 279.5 nm, via Jasco spectrophotometer, and TMC contents were calculated with previously drawn standard curve. Results were expressed as cumulative release percent of three replicates. Data obtained from this release study were fitted to the release kinetic models (zero order, first order, second order and Higuchi diffusion equations) to reveal the release kinetics. Correlation coefficients' values were compared for selection of the best release model that is best fits the data.

2.5 Physical characterization of niosomal vesicles 2.5.1 Particle size determination Particle size and poly dispersity index (PDI) were determined using dynamic light scattering integrated in a Zetasizer Nano-ZS (Malvern Instruments/ Herrenberg, Germany). Before measurement, all the samples were diluted with de-ionized water to generate appropriate scattering intensity. Three independent measurements were taken for each sample at the room temperature from the non-invasive back scatter technique where the detector lays at 173◦. 2.5.2 Morphological examination using Transmission Electron Microscopy (TEM) The morphology of the niosomal dispersions was examined using transmission electron microscopy (TEM). The freshly prepared sample was deposited onto the

surface of carbon coated copper grid (mesh size of 300) and left to dry to allow adhering of the niosomes to the carbon substrate. A drop of 1% aqueous solution of phosphotungstic acid dye was applied for staining and then grid subjected to air drying for 1-2 min, after the removal of excess dye by a piece of filter paper. The stained sample was then probed and visualized using TEM (FEI Techni, Holand) at operating voltage of 70 kV. Photographs were taken at suitable magnifications. 2.5.3 Thermal characterization using Differential Scanning Calorimetry (DSC) The thermal properties of weighed samples (3.5 - 4 mg) of the freeze dried niosomes were characterized using differential scanning calorimeter (DSC 200F3 Maia, Germany) equipped with liquid nitrogen cooling system. The measurements were carried out at a heating rate of 10 ºC/ min under flow of nitrogen gas with flow rate 25 ml/min to avoid sample oxidation. In order to provide the same thermal history, each sample was heated from room temperature to 200 ºC and rapidly cooled down to 25 ºC, then the DSC scan was recorded by heating from 25 to 200 ºC, using an empty aluminum pan as a reference.

2.6 In-vitro cellular uptake and cytotoxicity assay on breast cancer cells 2.6.1 Cell line Human breast cancer cell line (MCF-7) was grown in T-75 tissue culture treated flasks using colorless RPMI 1640 cell culture media supplemented with 10% heat inactivated fetal bovine serum(FBS) and penicillin/streptomycin mixture (0.1mg/ml). Cells were kept in CO2 incubator at 37°C under 5% CO2 and 95% air. Cells were kept in the exponential growth phase (sub-confluence) by sub-culturing the cells once reached 70% confluence with fresh media (Perillo et al., 2000). 2.6.2 Cellular uptake Cells were plated in 96- well plates, at a density of 5000 cells/well, in 200 µl of medium and allowed to attach for 24 hours prior to the initiation of experiments (Cosco et al., 2012). After adding free TMC and TMC loaded niosomes at a TMC concentration of 15µg/ml cells were incubated. Cells without any TMC added were used as a blank control. After 3, 6, and 24 hr incubation, the growth media taken and

the cells washed three times with PB. The collected media and washing PB were assayed for TMC concentration using HPLC-MS (Thermo scientific Accela, USA) using C18 column (Phenomenex 50×20 mm, 2.1 µm) and acetonitrile with 0.1% formic acid (9:1) as mobile phase (Dahmane et al., 2010). The calculated decrease in TMC /TMC niosomes concentration was used to figure out the cellular uptake. 2.6.3 Cytotoxicity assay The cytotoxicity of niosomal TMC was determined by Sulfo-RhodamineB (SRB) assay (Skehan et al., 1990). MCF-7 cells were seeded into 96-well culture plates, at a density of 10,000 cells/ well, and incubated for 24 hr at 37 °C to allow attachment of cells to the plates prior to use. Free TMC and TMC loaded niosomes were added to 96-well plate at 5, 12.5, 25 and 50 µg/ml TMC concentrations. Monolayer cells were incubated after drug treatment for 72 hr at 37 °C and in 5% CO2 incubator. After 72 hr incubation, cells were fixed with 10% trichloro acetic acid in PB for 30 min with caution to avoid dislodgement of cells before detachment. Plates then washed five times with PB and then 50 µl SRB stain solution were added to each well and placed on an orbital bench top shaker for 30 min. Excess stain was removed with five washes with 1% acetic acid and the attached stain was solubilized with 250µl of lysis buffer (10mM Tris- EDTA buffer by shaking for 30 minutes. From each well 200µl was transferred to a corresponding well of a 96-well plate and color absorbance was measured using ELISA micro-plate reader (Meter Tech. S960, Warminster, PA, USA) at 564 nm. A dose response curve was established by presenting different concentrations of TMC on the x-axis versus absorbencies on the y-axis. The half maximal cells growth inhibitory concentration (IC50%) was determined which is relative to the concentration that provides 50% cell viability. The cell viability was measured by calculating the relative absorbance for the treated cells compared to that of untreated controlled cells. 2.7 In-vivo anti-tumor effect on animal model 2.7.1 Animals and tumor cell implantation The animal study protocol was approved by the animal experimentation ethics committee of the Faculty of Pharmaceutical Science and Pharmaceutical Industries, Future University (Approval no: REC-FPSPI-2/13). Thirty six healthy non-breeding adult female Swiss Albino mice of average weight (20-25 g) were supplied from

National Research Center (NRC), Egypt. All mice were kept in Makrolon IV polycarbonate cage and allowed to a free access of normal tap water and regular Purina rodent diet pellet chow all over the period of the experiment. They maintained in a light controlled room at a temperature of 22°C and relative humidity of 55%. The mice were acclimatized to the animal house condition for two days prior to the experiment. To establish tumors in the Swiss Albino mice, Ehrlich carcinoma cell line was subcutaneously injected through 23-G needle into the right thigh of each mouse (0.2 ml/ 2-2.5×106 cells/ mouse) to facilitate tumor volume measurement (Silva et al., 2006). The palpable solid tumors were developed after 13 days with an average tumor solid mass volume > 100 mm3. After the mice developed palpable solid tumors, they were divided randomly into three groups (12 mice each). The first group was left as untreated control group and only received an intra-tumor injection of an isotonic saline. The second and third groups received an intra-tumor injection of TMC suspension and TMC niosomal suspension, respectively in a TMC dose 10 mg/kg body weight since the intra-tumor delivery of anticancer was considered a valid therapeutic strategy for treatment of cancer (Le et al., 2008; Arai et al., 2010; Xing et al., 2014). The first dose was received after 13 days post SEC induction (The first day of treatment) while the second dose was received after 19 days post SEC induction (The seventh day of treatment). Such multiple intra-tumor doses were reported in (Nakase et al., 2004; Yang et al., 2012; Van De Voort et al., 2013) 2.7.2 Tumor volume (V) and percentage tumor growth inhibition (% TGI) measurement Tumor volumes were recorded twice weekly from the first day of TMC injection till the day of mice scarification. This was attained by recording the two perpendicular diameter of the tumor mass; length (b) (large diameter in mm) and width (a) (small diameter in mm) of the developed solid tumor mass using digital caliber. Tumor volume was calculated as follow: (Abdin et al., 2014). Volume = (a2×b)/2

(2)

Drug efficiency is expressed as percentage tumor growth inhibition (TGI) which is calculated as: (Sancéau et al., 2002)

% TGI = 100- (T/C ×100)

(3)

Where T is the mean relative tumor volume (RTV) of the treated group and C is the RTV of control group. RTV of any group is calculated by the following equation: (Sancéau et al., 2002). RTV = Vx/Vi

(4)

Where Vx is the tumor volume at the end of the experiment (days of scarification) and Vi is the tumor volume at the start day of treatment. 2.7.3 In-vivo TMC quantification in plasma and tissue0 At the end point of the study (24 days after TMC treatment), the mice were sacrified and tumor, live and blood samples were taken and frozen at -80◦C for later TMC quantification. Blood samples were collected in heparinized Eppendorf tubes and plasma was separated by centrifugation at 4000 rpm for 10 min before freezing. One gram tissue samples of tumor and liver were weighed and rinsed immediately with saline and subjected to tissue homogenization in 5 ml of 50mM Tris-Hcl buffer using a probe tissue homogenizer at a rate 4000 rpm for 3 min. TMC in plasma, tumor and liver homogenate were extracted with equal volume of acetonitrile (Jain et al., 2011), followed by vortexing using the vortex mixer for 30 seconds. Finally, samples were centrifuged at a speed of 4000 rpm for 10 min to remove precipitated proteins. The supernatant was collected and filtered through 0.22 µm syringe filter, the filtrate was collected in an auto-sampler vial and the drug was assayed by HPLC-MS (Thermo scientific Accela, USA).

2.8 Statistical analysis Statistical analyses of data were performed using Graphpad Prism version 5.02 (Graphpad Software, Inc. La Jolla, USA) by using 2-way ANOVA followed by the Bonferroni post-tests. Data reported as means ± standard deviation (SD). Statistical differences between the groups were considered significant if the p value was < 0.05.

3. Results and discussion 3.1. Preparation and entrapment efficiency of TMC niosomes.

Our main goal was to formulate Tamoxifen citrate in a niosomal nanostructure delivery system as a smart local approach to overcome its toxicity challenge. Successfully, different TMC loaded niosomes were prepared with high entrapment efficiencies, Table one illustrated the entrapment efficiencies for these different niosomal preparations using different nonionic surfactants (Span 20, Span 40, Span 60 and Tween 20) with different molar ratio of cholesterol. As expected, the E. E of the TMC increased upon decreasing the hydrophilic character (represented by HLB value) of the used NIS. As shown, formulae were using Span 40 (HLB= 6.7) showed high entrapment efficiency up to 86.3± 2.4 (formula 3). This high entrapment was mainly due to the structural affinity between Tamoxifen (hydrophobic molecule) and the non-polar palmitate moiety (hydrophobic eleven C-H chain). Meanwhile, formulae were using Span 60 (HLB = 4.7) showed higher entrapment efficiency up to 92.3 ± 2.3 (formula 8). A rational speculation of this higher entrapment was the length of the C-H chain, which is fifteen alkyl chains in the stearate moiety of Span 60. Longer hydrophobic chain enabled more efficient trapping of the hydrophobic TMC during their assemblage into niosomes. However, Span 80 (HLB = 4.3) with the longest alkyl chain showed lower E. E than Span 40 and Span 60. This was basically due to introduction of double bonds in the oleate alkyl chain that made the chains more flexible to bend and rotate. This rotation will obviously increase the steric hindrance between the hydrophobic chains, and this make it more difficult to TMC molecules to adhere closely to oleate chain. In addition this hydrophobic hindrance will make the self-assembled niosomes more leaky, which also explains the low entrapment efficiency of the Span 80 formulation (Hao et al., 2002). Therefore, F8 composed of Span 60: CH (1: 1) and was considered the formula of choice as it had the highest E. E (92.3 ± 2.3) when compared to other formulae at any molar ratio. On the other hand, Tween 20 (HLB = 16.7) showed a remarkable lower E. E than Spans. This was clearly attributed to the predominant hydrophilic character of hydroxyl group and the ether oxygen in its molecular structure, and also understood from its high HLB value. At the same time this low EE also attributed to the higher gel to liquid phase transition temperature of Spans than Tween 20 ( Junyaprasert et al., 2012). Surfactants with higher phase transition temperature were more likely to be in the ordered gel form forming less leaky bi-layers than surfactants of lower phase

transition temperature which were more likely to be in a less ordered liquid form (Kumar and Rajeshwarrao, 2011; Shaker et al., 2013). Table 1 also showed that cholesterol had a profound effect on TMC entrapment in niosomes. The presence of cholesterol in the formulation of niosomes was deemed necessary for the formation and physical stability (i.e. keeping the assemblage integrity, vesicles size and dispersibility) of these nano-size vesicles. Hence, with all of the prepared formulations increasing CH content was associated with substantial increased the EE. This was mainly attributed to the increase in hydrophobicity (especially with higher HLB surfactant molecules) which increased the structural affinity of the bilayer membrane to TMC molecules. Along with the fact that increase in the intercalation of cholesterol in the bilayer membrane cause an augmentation in the membrane stability and a decreased in TMC leakage out of the prepared vesicles. Nonetheless with Spans surfactants the increment in the amount of incorporated CH above the equimolar ratio with NIS was accompanied by increase in the bilayer membrane rigidity. Rigid membrane assembles as large vesicles, so an increase the size of the prepared niosomes accompanied with decrease E.

E was shown.

Meanwhile it was reported that high CH content compete TMC for packing space within the bi-layer leading to exclusion of TMC from vesicles (Nasseri, 2005). 3.2 In-vitro TMC release Kinetics The release of TMC from 6 selected niosomal formulae was illustrated in figure 1. The formulae were selected on the basis of EE results and/or different composition. Formulae 2, 3, 7, 8 were selected on the basis of EE results, while F5 and F10 were selected to study the effect of span 40 and span 60 with high cholesterol content on drug release. Upon observing the release profile, we could reveal that 100% release of free TMC after 6 hr, while a remarkable controlled release of TMC from niosomes up to several days was observed. For all formulae, the cumulative release profile was apparently biphasic with an initial rapid release period which extended up to almost 4 hr followed by a slower release phase. The dumpiness in the release which occurred at the beginning can be regulated by the combination of diffuse mechanism in the outside layers of niosomes as well as emancipation of drug located on the outer most surface. In such circumstances, it was expected that the deport of the drug molecules

from the successional inner layer to the adjacent outer layers and then to the surface of the vesicle was also retarded (Hao et al., 2002; Hong et al., 2009). The release results also showed the effect of manipulating the niosomes composition by varying the used NIS and molar ratio of incorporated cholesterol on TMC release rate. As it appeared in figure 1 Span 60 niosomal formulae represented in F7, F8 and F10 exhibited slower TMC release when compared to Span 40 niosomal formulae, with the same molar cholesterol content, that represented in F2, F3 and F5, respectively. This expected release behavior could be explained by the structural affinity of TMC with alkyl chain of used surfactant. Having said that, the higher the chain length of used Span, the greater will be the affinity with TMC. Hence, the stearate moiety of Span 60 shows a higher affinity and lower release ability than the palmitate moiety of Span 40. Likewise, the resulting release data showed a significant dependence on the molar ratio of cholesterol. Increasing the cholesterol content decrease the amount of TMC released. Accordingly, formulae with high CH content (F5 and F10) exhibited slowest release rate due to decreasing niosomal membrane permeability that give more sustain of drug release (EL-Ridy et al., 2012). To elucidate on the TMC release kinetics, a linear regression of the release data was carried out. As shown in table 2, The TMC release data were analyzed mathematically according to: zero-order, first-order, second-order and Higuchi’s equations. From the regression coefficient results it showed a linear relation with Higuchi’s equation for example, the linear regression correlation coefficient was found to be equal to 0.992 for the release from formula F8. These confirm that the drug was released from niosomes by Higuchi diffusion controlled mechanism.

3.3 Physical characterization 3.3.1 Particle size analysis Dynamic laser scattering was performed to characterize the size of the selected TMC niosomal formula (F8). The formula showed small nano-vesicular size (212.2 ±57.5 nm). The particle size distribution for the formula was narrow with poly dispersity index (PDI) 0.521 as appear in table 3. The obtained data in table 3 showed that the hydrodynamic diameters of the prepared niosomes significantly increased

with increasing the C-H chain length of the used surfactant molecule. As such particle size for formula F3 was 89.9 ± 37.6 nm (PDI = 0.647) and for formula F8 was 212.2 ± 57.5 nm (PDI = 0.521). These results were in accordance to the fact mentioned that Span 60 (C18) has a longer alkyl chain length than Span 40 (C16), thus relative longer bi-layer length may increase the size of the vesicles (Uchegbu and Duncan, 1997). It was also really worth to mention that such nano-size ranged vesicles represent a good pharmaceutical carrier for passive targeting to cancer cells. Tamoxifen Citrate niosomes can easily permeate and retain inside the interstitial space causing accumulation in tumor tissue. Meanwhile, upon increasing the CH molar ratio the formula showed a significant increase in the mean diameter, as such formula F10 had mean size of 753.1 ± 154.7 nm (PDI = 0.453). This was mainly attributed to the increment in the width of the niosomal bi-layer as a result of CH embedment (Lee et al., 2005). Accordingly, to achieve a balanced correspondence between the niosomal size and TMC entrapment efficiency F8 was selected for further investigation. . 3.3.2 Transmission Electron Microscopy (TEM) Figure 2 showed the transmission electron microphotographs of formula F8, as a representative sample, that was composed of Span 60 with cholesterol in equimolar ratio. TEM employed here to picture directly the shape and size of TMC loaded niosomes. As occur in the images the niosomes appears as numerous scattered spherical dark stained nano vesicles with well-identified outline and core. 3.3.3 Differential Scanning Calorimetry (DSC) The thermal behavior of Span 60, Cholesterol, TMC, unloaded niosomes, and TMC loaded niosomes formula F8 are shown in Figure 3. The DSC thermogram of pure TMC showed a sharp endothermic peak at 145.8 °C (figure 3, C) corresponding to its melting point and indicating the crystalline nature of TMC with enthalpy fusion ∆H= 39.95 J/g (Shaker et al., 2014). The thermal analysis of Span 60 surfactant showed an endothermic peak at its melting point 59.32 °C with enthalpy fusion ∆H= 155.88 J/g (figure 4, A), whereas cholesterol showed also an endothermic peak at 51.56 °C with enthalpy fusion ∆H= 48.28 J/g (figure 3, B). Formulation of Span 60

and cholesterol as niosomes (formula F8) without incorporation of TMC showed a typical thermoset behavior no endothermic peaks (figure 3, D). Disappearance of the Span 60 and cholesterol peaks was a result of their assembly as a bilayer that loosens their crystalline pattern compared to those of cholesterol and Span 60, separately. At the same time, the incorporation of TMC into the niosomes diminished its crystallinity, as it was appeared in the thermogram of TMC niosomes formula F8 (figure 3, E). Knowing that the enthalpy ∆H is calculated in term of area under the peak and may be used for quantitative estimation of the crystallinity (Marin et al., 2002), so it indicated that the TMC was loaded in the niosomes through incorporation within the surfactant bilayer structure as an amorphous form. This could be thrives on the fact that the bilayer domain’s rigidity disrupted the regularity and the ability of the crystal lattice of the TMC to from regularly arrange.

3.4 In-vitro cellular uptake and cytotoxicity This test was performed to evaluate the cellular uptake behavior of niosomal TMC in comparison to free TMC at different time intervals (Chawla and Amiji, 2003). The efficiency for cellular uptake of TMC niosomes was determined after incubation with MCF-7 breast cancer cells. As it can be seen from the figure 4 (A), the cellular uptake depended on the incubation time for TMC and TMC niosomes. The cellular uptake of TMC niosomes formula F8 was significantly different than that of free TMC, at different time intervals up to 24 hr. It was found that the cumulative intracellular concentrations of free TMC after 3, 6 and 24 hr post incubation were 1.1%, 19.2% and 42% respectively, while niosomal TMC were 58.7%, 60.9% and 96% respectively. It can be concluded that the niosomes significantly interacted with the breast cancer cells as a fundamental and common characteristic of Nano size vesicles. This kind cell-nanoparticle interaction was usually associated with internalization through rapid non-specific phagocytosis (Hong et al., 2009). Furthermore, area under the curve (AUC) for the cellular uptake of free and niosomal TMC were 87.471 and 251.817 µg.hr/ml. Comparing these values, it was found that the overall cellular uptake of TMC niosomes was 2.8 fold greater than of free TMC.

To further explore the anticancer capability of these niosomes as pharmaceutical carrier for TMC, human breast cancer cells were incubated with TMC at different concentrations (5-50 µg/ml) and TMC loaded niosomes with equivalent drug concentration for 72 hr. Figure 4 (B) showed the cell viability for the MCF-7 cells after treatment with free TMC and TMC loaded niosomes (F8), using SRB assay method. The viability of the cancer cell to treatment depended on the concentration of TMC and TMC niosomes. With all of the used concentration, TMC niosomes exerted cytotoxicity to the cancer cells and was comparable to free TMC. Half maximal cells growth inhibitory concentration (IC50) for free TMC and TMC niosomes (F8) were 5.18, 8.78 µg/ml, respectively. However, the results demonstrated that free TMC and TMC niosomes showed no significant difference (similar cytotoxic effect) on cell viability after 72 hr of incubation. Even though this result that IC50 of free TMC was less than Niosomal TMC apparently expressed that TMC formulated into niosomes did not significantly improve the overall cytotoxic effect of TMC, TMC niosomes still considered better in anticancer candidacy for arresting the growth of MCF-7 cells. This could be explained based on only 62.2% TMC was released from loaded TMC niosomes after 3 days. Continuous release of TMC from niosomes was expected on the following days. These increasing amounts were expected to enhance cytotoxic activity despite the fact that the maximum period for all cytotoxicity studies is 3 days due to confluence and death of cells. The experiment could not be continued till 100% TMC release from F8 (Release data showed more than 6 days were required to release 100% of loaded TMC).

3.5 In-vivo anti-tumor effect The anti-tumor activity of TMC niosomes was evaluated comparatively to free TMC using SEC tumor bearing Swiss albino mice, as a valid model used frequently to investigate the chemotherapeutic efficacy for breast cancer (Silva et al., 2006). Tumor volume and tumor growth inhibition were used to measure the relative antitumor efficacy. Figure 5 (A) illustrated that the volume of the tumor increased rapidly for the mice were treated with isotonic saline (control group). However, for TMC treated groups, the antitumor activity of TMC niosomes and free TMC treatment was higher than the observed with control group. Lower tumor growth rates and lower tumor growth ratios were observed in case of mice treated with TMC and, especially in case

of TMC niosomes. As shown in figure 5 (A), little differences were found between TMC treated group and TMC niosomes treated group (tumor volume values were closer to each other) till day 29, where lowest values and significant differences in tumor volume and tumor growth rate were observed until the end of the experiment. Meantime, at the ends of the experiment the measured tumor growth inhibition for free TMC and TMC niosomes were 68.54 % and 84.04%, respectively. These results might indicate that the therapeutic efficacy and anti-tumor activity of TMC could be improved by entrapment into niosomes. It can't be overlooked here that the existence of free drug spikes that was associated with the in-vitro incubation was exclusively happen with free TMC, TMC incorporated within the niosomal bilayer slowly diffused and gradually released out in the surrounding microenvironment and consequently minimized the cytotoxicity. Accordingly the anticancer effect of TMC niosomes was almost the same as free TMC for in-vitro breast cancer cell line model. In-vivo however, the passive targeting ability and interstitial accumulation of TMC niosomes in the solid tumor potentiates its antitumor effects through localization and were anticipated to lower the TMC systemic side-effects.

To confirm tumor localization, TMC concentration in mice's blood, liver and solid tumor samples were determined at the end of the experiment (37 days post tumor induction) after intra tumor injection of F8 niosomes and free TMC suspension. As demonstrated in figure 5 (B), niosomal TMC significantly extended the retention of TMC in the solid tumor tissue. This was likely due to enhanced localization of niosomes into tumor cells (Mali, 2013). We had also quantified the concentration of TMC recovered from blood and liver to examine the distribution of TMC into different organs. Figure 5 (B) showed no significant difference in the TMC concentration when compared to free TMC. This was attributed to that a significant fraction of TMC niosomes injected around the tumor might have been forced out of the tumor because of the increased intra-tumor pressure generated by injection into solid tumor (Leet al., 2008). So as future perspective to prolong retention of a larger fraction of TMC inside tumor without diffusion out of it, it was best to disperse niosomal TMC into suitable biodegradable matrices, e.g. thermo-sensitive gel. Study for this point is still undergoing.

4. Conclusion This study explored the potential of TMC loaded niosomes as injectable delivery system for breast cancer therapy. Tamoxifen Citrate loading up to 92.3% was incorporated in the niosomal bilayer with the formulation of Spans 60 and cholesterol at equimolar ratio. In-vitro release results showed diffusion release behavior that was prolonged over 7 days. At the same time the release can be controlled through altering the alkyl chain length of used Spans and adjusting the molar ratio of incorporated cholesterol. The enhanced cellular uptake (2.8 fold) and cytotoxic activity with MCF7 breast cancer cell line as well as in-vivo tumor localization (passive targeting) and tumor regression activity combine to make it an attractive candidate for use as a nanosize delivery vehicle for delivery of TMC. A highlighted area of future investigation is expanding the application of TMC niosomes for incorporation inside a thermosensitive gel, to extend the release period and decrease the dosing rate during application for improving the patient compliance. The TMC loaded niosomes can be simply incorporated with biodegradable thermo-gelling agent to be injected inside the body using needles and catheters. Expanding this concept has a significant market potential and implements the full biomedical applications for TMC.

Acknowledgments The authors wish to thank both of Dr. Samia Shouman, National Cancer Institute, Cairo and Dr. Mohamed Nasr, Pharmaceutics Dept., Helwan University for their assistance.

Consent Not applicable.

Author Declaration The authors state no conflicts of interest and had received no funding for the research or in the preparation of this manuscript.

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Figure Captions

Figure 1: In-vitro release profiles of TMC from different TMC loaded niosomes formulae in PB containing 0.1% Tween80 at pH 7.4: (A) formulae using Span 40 (F2, F3, and F5) (B) formulae using Span 60 (F7, F8, and F10).Error bars represent the standard deviation of the mean of measurements from three samples

Figure 2: Representative transmission electron microphotographs of TMC loaded niosomes (formula F8). A; (scale bar = 500nm) B; (scale bar = 200nm).

Figure 3: DSC thermogram of Span 60 (A), cholesterol (B), TMC (C), F8 niosomes without TMC (D), and TMC niosomes (F8) (E).

Figure 4: (A) Time-dependent cellular uptake efficiency for TMC and TMC niosomes by MCF-7cells line. Each point represent the mean value ±SD (n= 3). (B) Cell viability of MCF-7 cell line treated with free TMC and TMC niosomal formula (F8) at different concentrations. Cell viability is calculated as a percentage of absorbance of treated cells over absorbance

of untreated cells. Each point represents the mean value ± SD (n= 3).

Figure 5: (A) Improved antitumor activity of TMC niosomes compare to that of free TMC and control (n=12). (B) In-vivo tissue distribution of the TMC in blood, liver as well as tumor tissue of Swiss Albino mice after intra tumor injection of free TMC and TMC niosomes (n = 6).

Span 40 Span 60 Span 80 Tween 20

Table 1: Composition and entrapment efficiency % (EE) of TMC loaded niosomal formulations using different non-ionic surfactant (NIS) with various molar ratios of cholesterol (CH).

Formula Used Molar ratio NIS of NIS: CH F1 3:1 F2 2:1 F3 1:1 F4 1:2 F5 1:3 F6 3:1 F7 2:1 F8 1:1 F9 1:2 F10 1:3 F11 3:1 F12 2:1 F13 1:1 F14 1:2 F15 1:3 F16 3:1 F17 2:1 F18 1:1 F19 1:2 F20 1:3 Regression Coefficient (R2)

Formula F2 F3 F5 F7 F8 F10

Zero 0.846 0.943 0.896 0.947 0.976 0.914

First 0.939 0.965 0.928 0.907 0.983 0.928

Second 0.894 0.834 0.952 0.617 0.894 0.941

Higuchi 0.986 0.983 0.967 0.981 0.992 0.973

EE% (Mean ± S.D) 45.5 ± 2.5 76.3 ± 5.1 86.3 ± 2.4 59.2 ± 4.9 37.4 ± 3.4 73.1 ± 3.7 80.5 ± 2.4 92.3 ± 2.3 56.5 ± 4.8 37.9 ± 4.1 10.6 ± 2.2 16.4 ± 3.3 58.9 ± 2.9 61.8 ± 4.3 36 ± 1.2 4.5 ± 0.4 7.6 ± 0.2 11.3 ± 0.1 29.4 ± 3 32.7 ± 2.5

Table 2: Release kinetics data of TMC from different niosomal formulae.

Formula

Vesicle diameter

Polydispersity index

(nm)

(PDI)

mean ± SD (n=3) F3

89.9 ± 37.6

0.647

F8

212.2 ± 57.5

0.521

F10

753.1 ± 154.7

0.453

Table 3: Vesicle size and polydispersity index of TMC loaded niosomal formulae F3; Span 40: CH (1: 1), F8; Span 60: CH (1: 1) and F10; Span 60: CH (1: 3).

A

B

B A

A B

MCF-7 celluar uptake efficiency

120

Free TMC TMC niosomes

100

80

60

40

20

0

3

24

6 Time (hours)

80

MCF-7 cell viability (%)

Free TMC TMC niosomes 60

40

20

0

5

12.5

25

Concentration (µg/ml)

50

A

Control (saline serum) Free TMC F8

3

Tumor Volume (mm )

14000 12000 10000 8000 6000 4000

2000

0

10

15

20

25

Time (days)

B

30

35

40

(ng/ml for blood and ng/gfor liver and tumor )

Tamxifencitrate concentration

3500

3000

Free TMC TMC niosomes

2500

2000

1500

1000

500

0

blood liver tumor