Design of colloidal drug carriers of celecoxib for use in treatment of breast cancer and leukemia

Materials Science & Engineering C 103 (2019) 109874

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Design of colloidal drug carriers of celecoxib for use in treatment of breast cancer and leukemia

T

Melike Ünera, , Gülgün Yenera, Mine Ergüvenb ⁎

a b

Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 34116 Istanbul, Turkey Istanbul Aydin University, Faculty of Medicine, Department of Medical Biochemistry, 34295 Istanbul, Turkey

ARTICLE INFO

ABSTRACT

Keywords: Acute promyelocytic leukemia Breast cancer Cancer therapy Celecoxib Solid lipid nanoparticles Nanostructured lipid carriers Nanoemulsions

Inflammation develops initiation and pathological process of cancer. Nanoscale drug carriers are required to be investigated for delivery of actives at cellular level for treatment of various cancer types. Solid lipid nanoparticles (CLX-SLN), nanostructured lipid carriers (CLX-NLC) and a nanoemulsion (CLX-NE) of celecoxib (CLX), a selective cyclooxygenase-2 inhibitor, were formulated for use in remedy of breast cancer and acute promyelocytic leukemia. The hot high pressure homogenization technique was employed to product formulations. Scanning electron micrographs were utilized for morphological characterization of formulations. Laser diffraction (LD), photon correlation spectroscopy (PCS) and differential scanning calorimetry (DSC) were used for examination of their physical stability by storing them at various temperatures. Drug release profiles of formulations were obtained. Their activity on cancer cells was investigated in the cell culture experiments. Stable formulations having homogenous size distribution were obtained below 200 nm with high drug payloads between 93.76% and 96.66%. Nanoparticles were ascertained to contribute controlled drug release. CLX-SLN induced the highest decrease in numbers of human breast cancer and human acute promyelocytic leukemia cells through the activation of the cell death cascades especially apoptosis in comparison to CLX-NLC, CLX-NE and the pure CLX application (p < 0.05). Nanoformulations of CLX optimized in this study were found to have various advantages expected from sophisticated drug delivery systems in order to achieve higher CLX efficiency at cellular level. Thus, they are able to be administered efficaciously alone and in combination therapies in remedy of breast cancer and acute promyelocytic leukemia.

1. Introduction Chronic inflammation plays a critical role in initiation and invasion of cancer [1]. Non-steroidal anti-inflammatory drugs (NSAIDs) restrain the nuclear transcription factor, NF-κB which is found overexpressed in cancer cells. NF-κB modulates the expression of enzymes and proteins such as cyclooxygenase-2 (COX-2) and cyclin D1 associated with inflammation and cellular proliferation [2]. Thus, COX-2 suppression leads to a substantial alleviation in the risk of cancer formation/progression and metastasis. Celecoxib (CLX) is a selective COX-2 inhibitor, which lays out a considerable anti-cancer activity in different cancer types including breast cancer and leukemia [3–5]. It introduces a new approach for impeding cancer formation and for improving efficacy or further eluding drug resistance alone or in combination with chemotherapy. Administration of actives in colloidal drug carrier systems has been aimed by various research groups. To enhance bioavailability of actives in cancer therapy can be possible by improving their tissue

⁎

distribution with their incorporation into drug delivery systems and subsequently, facilitating internalization of drug carrier systems by tumor cells [6,7]. In other words, actives can be applied with enhanced efficacy and minimized side effects by loading them into colloidal drug carriers. Surface modification of colloidal drug carriers is expected in order to protect them against phagocytic uptake by macrophages of the reticuloendothelial system (RES) - mononuclear phagocytic system. PEGylation is the most commonly used way to obstruct adsorption of opsonins (e.g., Complement C3b, fibronectin and immunoglobulins) on to nanocarriers and afterwards, instantaneous uptake of nanoparticles by macrophages of RES [7,8]. SLN, NLC and NE, which are outstanding delivery systems, are researched extensively for delivery of anti-neoplastic agents, biological materials and NSAIDs at cellular level for treatment of various cancer types [8–10]. Their surface modification is also compulsory to prolong their stay in the systemic circulation. Because, surface characteristics and physicochemical properties of SLN, NLC and NE such as particle/

Corresponding author. E-mail address: [email protected] (M. Üner).

https://doi.org/10.1016/j.msec.2019.109874 Received 29 October 2018; Received in revised form 23 March 2019; Accepted 7 June 2019 Available online 08 June 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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droplet size and surface hydrophobicity assign whether opsonization process occurs or not. Hydrophilic residues of polyethylene glycol (PEG) molecules front to the aqueous phase of the colloidal dispersion when lipophilic residues settle on the lipophilic nanoparticle surface by formation of a shell around it in PEGylation process. PEGylated NEs also introduce a beneficial alternative for transport of actives at cellular level [11]. PEG coating provides a sufficient strength at surface of droplets and existence of droplets can be prolonged in the systemic circulation. PEGylated SLN, NLC and NE of CLX were attempted to formulate for treatment of breast cancer and acute promyelocytic leukemia in this study. For this purpose, formulation parameters were intensively investigated and data obtained were evaluated. Particle/droplet morphology of CLX-SLN, CLX-NLC and CLX-NE was imaged by SEM. Physical stability of formulations was monitored by storing them under various thermal conditions [25 °C ± 2 (room temperature, RT), 4 °C ± 2 and 40 ± 2 °C]. While particle/droplet size of formulations was measured by LD and PCS, thermal behaviors of nanoparticles were investigated by differential DSC. In vitro drug release from CLX-SLN, CLX-NLC and CLX-NE was investigated using the dialysis bag technique. Then, anti-cancer efficiency of formulations was investigated on human breast cancer cells (MCF-7) and human acute promyelocytic leukemia cells (HL-60) comparing to the pure CLX. All applications were evaluated by indices of cell proliferation, apoptosis and cell viability, apoptotic protein levels as caspase-3, Bcl-2-associated X protein (bax), anti-apoptotic (survival/resistant) protein levels as COX-2, protein kinase B (AKT), phospho (p)-AKT, midkine (MK) and B-cell lymphoma 2 (bcl-2), drug efflux proteins as adenosine triphosphate ATP-binding cassette (ABCG2) and P-glycoprotein (P-gp).

2.3. Preparation of the formulations Placebo (P-SLN, P-NLC and P-NE) and CLX loaded formulations (CLX-SLN, CLX-NLC and CLX-NE) were prepared using high pressure homogenization at 85 ± 1 °C [13]. Production process of formulations was repeated 3 times for confirmation of the reproducibility of the method. For this purpose, a hot aqueous surfactant solution (Cremophor® EL) was added to the melted lipid (Precirol® ATO 5) and to the melted lipid:CLX mixture (9.9 Precirol® ATO 5:0.1 CLX) for production of P-SLN and CLX-SLN, respectively. Ratios of the lipophilic phase and surfactant were 10% (w/w) and 1.5% (w/w) in formulations. The aqueous phase was containing 2.5% (w/w) glycerol for providing the isotonicity of dispersions. Coarse emulsions were obtained stirring with an ultraturrax (IKA, Germany) at 20.000 rpm for 1 min. They were homogenized in a double-stage APV-2000 high pressure homogenizer (SPX Flow Technology, Poland) respectively at 50 bar and 1500 bar pressures for 3 cycles. Hot emulsions in nanometer size range were sealed in transparent vials. For preparing P-NLC, 3% liquid lipid (Miglyol® 812) was added to the melted lipophilic phase changing with the equal amount of Precirol® ATO 5. 0.1% CLX was incorporated to the lipophilic phase changing with the equal amount of Miglyol® 812 for producing CLXNLC. P-NE and CLX-NE were prepared using Miglyol® 812 instead of Precirol® ATO 5 in P-SLN and CLX-SLN by the same technique under the same conditions. 2.4. SEM analysis A scanning electron microscope (Jeol NeoScope, JCM-5000, USA) was used for imaging CLX-SLN, CLX-NLC and CLX-NE [13]. 2 μL sample was placed on a glass surface and dried in an oven at 40 ± 1 °C. Then, it was coated with gold using an ion sputter for 3–4 min. The sample was examined at an accelerating voltage of 15 kV.

2. Materials and methods 2.1. Materials In this study, analytical grade chemicals were used. Celecoxib (CLX) (ARYL SA Productos Quimicos, Argentina) was thankfully donated by Kale Kimya Group in Istanbul. Precirol® ATO 5 (GatteFossé, France) and Miglyol® 812 (Sasol, South Africa) were kindly presented by B'IOTA Laboratories (Turkey). Cremophor® EL and Spectra/Por® dialysis membrane (MWCO: 3.500) were purchased from Sigma (Germany) and Spectrum Laboratories Inc. (USA), respectively. MCF-7 human breast cancer (HTB-22™) and HL-60 human acute promyelocytic leukemia (CCL-240™) cell lines were acquired from the American Type Culture Collection (Wesel, Germany). Bax, bcl-2 and COX-2 were purchased from IBL America (USA). Akt and p-akt were purchased from Invitrogen Lifesciences (UK). Caspase-3 were purchased from Sigma-Aldrich (Germany). MK, adenosine triphosphate ATP-binding cassette (ABCG-2) and P-gp were acquired from USCN Life Science Inc. (USA).

2.5. Determination of drug content in SLN, NLC and NE The entrapment efficiency (EE) and loading capacity (LC) (%) of formulations were ascertained analyzing the free drug amount in their dispersion medium as reported in an earlier study [14]. Non-encapsulated CLX was separated by filtration-centrifugation using centrifugal filter units containing Amicon® Ultra-4 tubes, (Merck, Germany) at 5000 rpm (Heraeus Biofuge Primo R Centrifuge, Thermo Fisher Scientific, Germany) for 30 min. Proper amounts of supernatants were taken and diluted to 10 mL with a water:propylene glycol:ethanol mixture (50:25:25, v/v/v). The quantities of the free drug in supernatants were analyzed by high performance liquid chromatography (HPLC) as reported in our earlier study [13]. The analytical quantification method was validated according to the instructions of ICH Q2 (R1) guideline, the text on validation of analytical procedures prior to the analysis [15]. EE and LC were attained using Eqs. (1) and (2). This study was repeated 6 times for each formulation.

2.2. Lipid screening Lipid screening experiment was accomplished according to a method descibed by Pardeike et al. [12]. Increasing amounts of the drug [CLX:lipid (0.3:9.7, 0.4:9.6 and 0.5:9.5, w/w)] were included to lipids to determine solid and liquid lipids dissolving the highest concentration of CLX. Drug:lipid mixtures were agitated using a shaking water bath (Daihan Scientific Wisebath®, South Korea) set to constant 250 rpm agitation and 85 ± 1 °C for 2 h. They were observed using a light microscope with 200-times magnification (Carl Zeiss Axio Lab.A1, Carl Zeiss, Germany) equipped with crossed polarizers and scored. To investigate the recrystallization of CLX in solid and liquid lipids, mixtures were also observed using the light microscope after 24 h. In addition to the solubility of CLX in lipids, miscibility of all solid and liquid lipids was evaluated under the same conditions. This study was conducted in triplicate.

EE =

initial amount of drug free drug x100 initial amount of drug

(1)

LC =

initial amount of drug free drug x100 amount of lipid used

(2)

2.6. Physical stability assessment Placebo and drug loaded formulations in clear silanized glass vials were stored at RT, 4 ± 2 °C and 40 ± 2 °C in the dark [16,17]. Samples were collected on the production date of formulations and subsequently on the 7th, 30th, 60th, 90th and 180 days of storage. Drug expulsion from nanoparticles/droplets by time was investigated by the 2

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method used for determination of EE. DSC, PCS and LD assessments were also carried on samples.

amino acid solution, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin, 1.5 g/L sodium bicarbonate in a humidified atmosphere at 37 °C in 5% CO2. MCF-7 cells treated with tyripsinethylenediaminetetraacetic acid (EDTA), washed and resuspended in DMEM-F12 for experiments [24]. Freshly prepared nanoformulations (CLX-SLN, CLX-NLC and CLXNE) were passed through 0.2 μm polyethersulfone filters (Minisart®, Sartorius GmbH, Germany) and transferred to sterile 2 mL stoppered test tubes prior to cell culture experiments. Formulations equal to 10 μM CLX were applied to all cell lines. Groups were determined as the control group (untreated), the pure CLX group and groups of formulations. All applications were evaluated by indices of the cell proliferation, apoptosis and cell viability, apoptotic protein levels as caspase-3, bax, anti-apoptotic protein levels as COX-2, AKT, p-AKT, MK and bcl-2, drug efflux proteins as ABCG2 and P-gp for 48 h. Cell culture experiments were conducted in triplicate.

2.7. DSC analysis For determination of thermal behaviors of the solid lipid in nanoparticles and the drug:excipient interaction, DSC analysis was carried out on the pure solid lipid and nanoparticle formulations [13,18]. For this intent, 40 μL dispersion equal to 2–4 mg solid lipid was precisely weighed in standard sealed aluminium pans of the apparatus (Perkin Elmer Jade DSC, USA). Sealed pans were placed in the sample chamber of the apparatus. An empty sealed pan was also used as the reference in experiments. Pans were then heated from 20 °C to 180 °C with a heating rate of 10 °C/min flushing with 30 mL N2/min. Melting peaks and enthalpies of samples were calculated using the Pyris™ Software. Crystalline state of the solid lipid in nanoparticles was investigated by calculating its crystallinity indice (CI).

2.10.1. Cell proliferation index Cells stained with trypan blue were counted with an automated cell counter (Luna-II™ from Logos Biosystems, Annandale, USA) [23–25].

2.8. Particle and droplet size measurements PCS (Zetasizer Nanoseries, Nano-ZS, Malvern Instruments, UK) and LD methods (Mastersizer 2000 laser diffractometer equipped with the Hydro 2000MU wet sample dispersion unit, Malvern Instruments, UK) were used for measuring particle and droplet sizes of formulations [19]. Samples were diluted with water at a certain ratio before measurements. Polydispersity index (PI) values presented the width of particle size distribution in PCS experiments. Particle and droplet size distribution of samples were detected by LD that d(0.1), d(0.5) and d(0.9) were obtained as the volumetric distribution data. The advantage of the Mie theory was taken for transforming intensity distribution data obtained to volume in LD measurements. Particle and droplet size measurements were repeated in triplicate.

2.10.2. Cell viability index The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) reduction assay was performed using the cell proliferation kit I MTT (Roche, Mannheim, Germany) according to the manufacturer's protocol with some minor modifications [23]. Briefly, cells at a logarithmic growth period were put into 96-well plates at a density of 5 × 103 per well and incubated with treatment agents for 48 h. At the end of the 24th and 48th h, 10 μL MTT (5 mg/mL) was added to each well. Supernatants were discarded, cells were incubated with 150 μL dimethyl sulfoxide (DMSO) in a dark room for 10 min for complete dissolution of crystalline substances. The optical density (OD) of each well was measured using a microplate reader. The inhibition rate of cell proliferation (%) was calculated as 100% [(OD value in the control group)-(OD value in the experimental group)]/(OD value in the control group).

2.9. In vitro drug release The dialysis bag technique was used to attain drug release profiles of CLX-SLN, CLX-NLC and CLX-NE. 1 g samples were initially filled into regenerated cellulose dialysis bags (Spectra/Por®, MWCO: 3.500 Da) and both ends of bags were closed tightly [13,20]. Bags were immersed into flasks containing 220 mL water:propylene glycol:ethanol mixture (50:25:25) as the release medium. Flasks were embedded in the water bath adjusted to constant 60 rpm agitation at 37 ± 0.5 °C temperature. “Sink condition” was provided for avoiding interference of the drug solubility in the release medium. In the meantime, 0.1% (w/v) CLX solution in water containing 2.5% (w/w) glycerol was also prepared and tested as the control. 1 mL samples were taken from flasks at predetermined time intervals for 48 h and amounts of the drug were determined in samples by HPLC. This study was replicated in 6 times. Drug release profiles of formulations were appraised considering zero order, first order and Higuchi square-root kinetic models [21]. Korsmeyer-Peppas kinetic model was also referred for confirmation of release mechanisms. For this purpose, “n” values contributed accurate knowledge for more than one type of release mechanisms comprised [22].

2.10.3. Apoptotic index The apoptotic index was determined by the flow cytometric annexin-V-fluorescein isothiocyanate/propidium iodide (annexin-V-FITC/ PI) staining following the instructions of commercial kit (BD Pharmingen, San Diego, CA, USA) applying some minor modifications [25]. After harvesting cells every 24 h, they were washed twice with phosphate buffered saline and resuspended by binding buffer containing 0.01 M 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), 0.14 mM NaCl and 2.5 mM CaCl2. A cell suspension (1 × 105 cells in 100 μL) in a binding buffer was incubated with 5 μL of FITClabeled annexin V dye and PI fluorescence for 15 min in the dark at RT. Viable, apoptotic and dead cell rates were measured by a flow cytometer (BD FACS/Calibur; BD Pharmingen, San Diego, USA) and analyzed with CellQuest and WinMDI programs (BD Pharmingen, San Diego, USA). 2.10.4. Apoptotic and anti-apoptotic protein levels Apoptotic and anti-apoptotic (survival/resistance) protein levels were determined by using commercially available colorimetric, enzyme-linked immunosorbent assay (ELISA) kits. All tests were performed by the same person according to the instructions of manufacturer with some minor modifications [25]. Bax, bcl-2, COX-2, akt, pakt, caspase-3, MK, ABCG2, P-gp were used for tests. Protein quantification of cell lysates was made by using the bicinchoninic acid (BCA) assay. Samples, standards and controls were incubated in a target specific antibody-coated wells about 1–2 h. Following the incubation, primary antibody was applied about 30–60 min. After that, wells were incubated with a secondary antibody with streptavidin conjugated to Horseradish peroxidase (HRP) about 20–30 min. At the last step, the

2.10. Cell culture Human acute myeloid leukemia (AML) cells as HL-60 (CCL-240™), non-adherent cells, were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) medium plus 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM non-essential amino acid solution, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin, 1.5 g/L sodium bicarbonate in a humidified atmosphere at 37 °C in 5% CO2 [23]. The human breast cancer cell line as MCF-7 (HTB-22™), adherent cells, is cultured in DMEM-12 with 10% heat inactivated FBS, 1 mM sodium pyruvate, 0.1 mM non-essential 3

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chromogen substrate 3′,3′,5,5′-tetramethyl benzidine (TMB) was applied about 3–20 min and when the blue color was detected as a sign of reaction positivity, the reaction was finished by the stop solution containing hydrocloric or sulfuric acids. ODs were measured at 405 nm and 450 nm (the reference wavelength: 690 nm) in 2–5 min by a microplate reader (Biotek, Bad Friedrichshall, Germany) and data were recorded.

approximately 250 nm particle and droplet size [13]. Thus, we performed this study with Precirol® ATO 5 and Miglyol® 812 for production of CLX-SLN, CLX-NLC and CLX-NE. 3.2. SEM analysis and particle/droplet size of formulations SEM images contributed characterization of morphology of CLXSLN, CLX-NLC and CLX-NE. They also provided us information on particle/droplet size of formulations (Fig. 1). Datum obtained from PCS and LD analysis were confirmed to be consistent with SEM datum. Mean particle/droplet size of CLX-SLN, CLX-NLC and CLX-NE was found as 119 nm (0.136 PI), 99 nm (0.201 PI) and 93 nm (0.149 PI) in PCS experiments, respectively (Fig. 3). d(0.5) as a parameter for the volume distribution of formulations was obtained as 194 nm, 192 nm and 193 nm in LD experiments (Fig. 2). It was confirmed that dispersions were free of microparticles and drug crystals.

2.10.5. Statistical analysis One-way analysis of variance (ANOVA) and Tukey's multiple range tests (GraphPad Prism software) were used to identify difference between drug release profiles of formulations. Data were evaluated at 0.05 as the level of significance. All statistical analyses for cell culture experiments were performed using SPSS Statistics 22.0 (IBM, Chicago, IL, USA) and graphs were drawn by using Microsoft Excel 2017. Independent Student-t-test was used for statistical analyses. All data were shown as means ± standard error means (SEM) and p < 0.05 was considered to be significant.

3.3. Determination of drug content in SLN, NLC and NE

3. Results

EE of CLX-SLN, CLX-NLC and CLX-NE was found to be 93.76 ± 0.21%, 95.43 ± 0.88% and 96.66 ± 0.4%, respectively. LC of formulations was 0.947 ± 0.002%, 0.964 ± 0.009% and 0.976 ± 0.004% in the same order.

3.1. Preparation of the formulations CLX was found to be soluble in Precirol® ATO 5, 1-octadecanol, Miglyol® 812 and oleic acid since existence of any crystals was not observed in drug:lipid mixtures (Table 1). Then, further experiments were detailed in the study. We also saw that Miglyol® 812 was physically compatible with Precirol® ATO 5. PCS measurements confirmed that we could obtain nanoparticles lower than 150 nm in average using Cremophor® EL (PEG-35 castor oil) as a PEG derivative surface active agent (see the next section). On the other hand, 1-octadecanol and oleic acid were compatible with Tween® 80 for giving SLN, NLC and NE with

3.4. Physical stability assessment Changes in the drug content of CLX-SLN, CLX-NLC and CLX-NE after 6 months of storage at different conditions are presented in Fig. 3. DSC thermograms of the pure drug (CLX), bulk solid lipid (Precirol® ATO5) and nanoparticles (P-SLN, P-NLC, CLX-SLN and CLX-NLC) are presented in Fig. 4. The melting point and enthalpy of the solid lipid were determined to decrease in nanoparticles. In other words polymorphic transition of the solid lipid in nanoparticles was found to be slower compared to the bulk lipid (Table 2). Polymorphic transition of the solid lipid to its more stable β-polymorph was confirmed to occur only in the bulk solid lipid [26,27]. On the other hand, the polymorphic transition process of the solid lipid slightly accelerated by the presence of the drug in the carrier. The storage temperature was observed to affect the polymorphic transition behavior of the solid lipid in nanoparticles for 6 months of storage. Because, decrease in the storage temperature was confirmed to accelerate the transition of the solid lipid to its more stable polymorph as can be seen in Table 2. The melting point of CLX was found as 166.07 °C (Fig. 4). The area under the curve and height of the drug peak were also detected to diminish in CLX-SLN and CLX-NLC compared to the pure drug. The place of the drug peak was confirmed to shift in nanoparticles. Mean particle/droplet size of PSLN, PNLC and PNE was measured by PCS as 121 nm (0.132 PI), 100 nm (0.126 PI) and 96 nm (0.119 PI), respectively. It was ascertained that the addition of the liquid lipid to the lipophilic phase did not significantly altered the particle size of PSLN. And, drug incorporation to formulations did not cause significant changes in their particle/droplet size (Figs. 2 and 5). CLX-SLN, CLXNLC and CLX-NE had homogeneous particle/droplet size distribution as can be seen in LD profiles. All formulations were further stored at various thermal conditions to demonstrate their physical stability since alterations in particle/ droplet size would be indicative and informative parameters for the stability of colloidal drug delivery systems during storage. While agglomeration occurred in CLX-SLN at 4 ± 2 °C and 40 ± 2 °C, it occurred in CLX-NLC at 4 ± 2 °C (Figs. 5 and 6). On the other hand, the droplet size of CLX-NE slightly changed during storage under all thermal conditions for 6 months of storage.

Table 1 Evaluation of solid and liquid lipids and their ability to dissolve CLX. CLX:Lipid 0.3:9.7

0.4:9.6

0.5:9.5

Solid lipids 1-octadecanol 1-tetradecanol Apifil® Carnauba wax Compritol 888® ATO Cutina® CP Dynasan® 118 Emulcire® 61 WL 2659 Gelucire® 33/01 Gelucire® 44/14 Gelucire® 50/13 Imwitor® 191 Imwitor® 900 Labrafil-M2130 CS Monosteol® Nafol® 16 18S Precirol® ATO5 Stearic acid Tego Alkanol® 16 Tego Alkanol® 1618 TegoCare® 450

+ (+) − − (+) − − (+) (+) (+) (+) − (+) (+) (+) − + − − − −

+ − − − − − − (+) − − − − − − − − + − − − −

+ − − − − − − (+) − − − − − − − − + − − − −

Liquid lipids Cetiol® Cetiol® OE Eutanol® Isopropylmyristate Labrafil® M1944 CS Miglyol® 812 Oleic acid

− − (+) − − + +

− − − − − + +

− − − − − + +

3.5. In vitro drug release

+, dissolved; (+), dissolved in heat but recrystallization at room temperature; −, not dissolved.

In the first 8 h CLX-NE was found to have the highest drug release 4

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Fig. 1. SEM images of CLX-SLN (a), CLX-NLC (b) and CLX-NE (c).

48th h: 1010000 ± 333, p < 0.01), (33 ± 2% and 37 ± 1% decrease, respectively)] for 48 h. The CLX-NE group induced the lowest decrease among formulations [(the 24th h: 982000 ± 577, p < 0.05; the 48th h: 1400000 ± 8819, p < 0.05), (14 ± 2% and 13 ± 0% decrease, respectively)]. CLX induced the lowest decrease in the cell number [(the 24th h: 964000 ± 5200, p < 0.05; the 48th h: 1530000 ± 1270, p < 0.05), (15 ± 2% and 5 ± 1% decrease, respectively)] in comparison to formulations.

rate (8.437 μg/h) while CLX-SLN and CLX-NLC displayed sustained drug release (Fig. 7). CLX-NE released 92.51% of CLX within 8 h. 100% drug release finalized up to 48 h. Release rate increased as the amount of the liquid lipid in the lipophilic phase increased in formulations. Drug release rate of CLX-SLN and CLX-NLC was calculated as 0.833 μg/ h and 1.350 μg/h, respectively. It was found out that CLX-NE released 100% of CLX at the end of 48th h while CLX-NLC and CLX-SLN released 69.46% and 40.38% of the drug, respectively (p < 0.05). While 100% of the drug release was occurred in the control CLX solution within 1 h, it was confirmed that formulations displayed the sustained drug release. The drug release profile of CLX-NE displayed a biphasic shape. The first phase of the release profile was found out to indicate 80.21 ± 0.36% of CLX release within 4 h. Then, 100% of CLX release was over up to the 48th h in the second phase. This can be ascribed to the barrier function of the film layer formed by the adsorption of surfactant molecules by droplets. The drug release profile of CLX-NE could be the best characterized by Korsmeyer–Peppas model (Table 3). Korsmeyer-Peppas model indicated Fickian diffusion release (case I diffusional) as the dominant mechanism for CLX-NE since “n” value was 0.44, lower than 0.45 (Table 3). Whereas, drug release from CLX-SLN and CLX-NLC followed square root drug release model (Higuchi model) with nonFickian anomalous transport as Korsmeyer-Peppas model indicated.

3.6.2. Cell viability index All groups induced decrease in the cell viability in comparison to the control group for 48 h (the 24th h: 97 ± 1%, the 48th h: 98 ± 1%). CLX-SLN induced the highest decrease in the cell viability (the 24th h: 56 ± 2%, p < 0.01; the 48th h: 23 ± 4%, p < 0.001). The lowest decrease was determined in the CLX-NE group (the 24th h: 82 ± 2%, p < 0.05; the 48th h: 48 ± 3%, p < 0.001). CLX-NLC led to mild decrease among formulations (the 24th h: 64 ± 1%, p < 0.01; the 48th h: 59 ± 3%, p < 0.05). CLX induced the lowest decrease in the cell viability (the 24th h: 92 ± 2%, p < 0.05; the 48th h: 93 ± 4%, p < 0.05) in comparison to formulations. 3.6.3. Apoptotic index Formulations and CLX increased the apoptotic index in comparison to the control group for 48 h (the 24th h: 2 ± 0%, the 48th h: 3 ± 1%) (Fig. 8A). Compared to the control group, the highest increase in the apoptotic index was determined in the CLX-SLN group as 36 ± 2% at the 24 th h (p < 0.01) and 72 ± 0% at the 48 th h (p < 0.001) and the lowest increase was detected in the CLX-NE group (the 24th h: 16 ± 1%, p < 0.05; the 48th h: 16 ± 3%, p < 0.05). The CLX-NLC group induced mild increase (the 24th h: 26 ± 1%, p < 0.05; the 48th h: 21 ± 2%, p < 0.05) among formulations. CLX led to the lowest increase in the apoptotic index (the 24th h:6 ± 2%, p < 0.05; the 48th h: 7 ± 3%, p < 0.05) in comparison to formulations.

3.6. Effects of different CLX applications on MCF-7 cells 3.6.1. Cell proliferation index All drug applications decreased cell numbers in comparison to the control group for 48 h (the 24th h: 1136667 ± 27,285, the 48th h: 1610000 ± 8819). For formulations, the highest decrease in cell number was determined at CLX-SLN [(the 24th h: 670000 ± 577, p < 0.05; the 48th h: 395000 ± 1155, p < 0.001), (41 ± 2% and 7 ± 1% decrease, respectively)] for 48 h. Mild decrease was detected at the CLX-NLC group [(the 24th h: 764000 ± 1155, p < 0.01; the 5

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Fig. 2. Particle and droplet size distributions of CLX-SLN, CLX-NLC and CLX-NE after their production (Laser Diffraction (LD) data: volume distribution).

Fig. 3. CLX content (%) of CLX-SLN, CLX-NLC and CLX-NE (PD: production date) (* indicates a statistically significant difference in drug amounts between PD and after 6 months of storage at various temperatures, p < 0.05).

3.6.4. Apoptotic and anti-apoptotic protein levels Effects of all applications on apoptotic and anti-apoptotic protein levels were clearly seen in Fig. 8B. Effects of formulations were compared with CLX. CLX induced the highest increase in COX-2, MK, Bcl-2, ABCG2 and P-gp levels, and p-AKT/AKT ratio in comparison to

Fig. 4. DSC thermograms of the pure drug, bulk solid lipid (Precirol® ATO 5) and nanoparticles (P-SLN, P-NLC, CLX-SLN and CLX-NLC) after production. 6

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Table 2 DSC parameters of the bulk solid lipid (Precirol® ATO5), placebo (P-SLN and PNLC) and CLX loaded lipid nanoparticles (CLX-SLN and CLX-NLC) after 6 months of storage at various temperatures.

Bulk Precirol® ATO5 Formulations P-SLN RT 4 °C 40 °C CLX-SLN RT 4 °C 40 °C P-NLC RT 4 °C 40 °C CLX-NLC RT 4 °C 40 °C

ΔH (J/g)

MP (°C)

CI (%)

241.27

61.17

100

10.03 11.52 7.64 10.26 12.43 7.56 5.37 5.90 4.71 5.77 6.20 5.04

59.57 59.74 58.24 59.69 59.72 57.86 57.37 58.51 56.93 56.89 58.62 55.76

48.67 55.90 37.07 50.29 60.93 37.06 37.23 40.90 32.65 40.58 43.60 35.45

Fig. 6. Mean diameter (nm) and polydispersity index (PI) of CLX-SLN, CLX-NLC and CLX-NE during 6 months of storage (PD: production date as open bars) (** indicates a statistically significant difference in data between PD and after 6 months of storage at various temperatures, p < 0.01).

lowest decrease was induced by CLX in comparison to formulations [(the 24th h: 840500, p < 0.05; the 48th h: 1390000, p < 0.05), (7 ± 0% and 5 ± 0% decrease, respectively)].

RT: room temperature, ΔH: melting enthalpy, MP: melting point, CI: crystallinity indice.

3.7.2. Cell viability index Formulations and CLX induced decrease in the cell viability in comparison to the control group for 48 h (the 24th h: 98 ± 1%, the 48th h: 99 ± 1%). CLX-SLN induced the highest decrease (the 24th h: 63 ± 3%, p < 0.01; the 48th h: 7 ± 4%; p < 0.0001). The lowest decrease was detected at the CLX-NE group (the 24th h: 85 ± 1%, p < 0.05; the 48th h: 30 ± 1%, p < 0.001). The CLX-NLC group led to the mild decrease (the 24th h: 83 ± 0%, p < 0.05; the 48th h: 28 ± 1%, p < 0.001). The lowest decrease in the cell viability among all applications was detected at the CLX group (the 24th h: 91 ± 0%, p < 0.05; the 48th h: 94 ± 2%, p < 0.05).

formulations, however the lowest increase in caspase-3 and bax levels was detected at this group. The highest increase in caspase-3 (p < 0.0000001) and bax (p < 0.0000001) levels was detected at the CLX-SLN group and the lowest increase was induced by the CLX-NE group (p < 0.05 for caspase 3, p < 0.05 for bax). The CLX-SLN group led to the highest decrease in COX-2 levels (p < 0.00001), the lowest decrease was detected at the CLX-NLC group (p < 0.001). The CLX-NE group led to the highest decrease in Bcl-2 (p < 0.001) and MK (p < 0.001) levels, the lowest decrease at these levels was detected in the CLX-SLN group (p < 0.01 for bcl-2, p < 0.01 for MK). The p-AKT/ AKT ratio was decreased efficiently by CLX-NLC (p < 0.0001), CLX-NE (p < 0.0001) and CLX-SLN (p < 0.01), respectively. The CLX-SLN group induced the highest decrease in ABCG2 (p < 0.00001) and P-gp (p < 0.00001) levels, the lowest decrease was detected in the CLX-NE group (p < 0.01 for ABCG2, p < 0.01 for P-gp).

3.7.3. Apoptotic index All groups increased apoptotic cell rates in comparison to the control group for 48 h (the 24th h: 3 ± 0%, the 48th h: 4 ± 1%) (Fig. 8A). The highest increase was determined in the CLX-SLN group which has apoptotic index values as 28 ± 0% at the 24 th h (p < 0.0001) and 87 ± 1% at the 48th h (p < 0.001) compared to the control group. The CLX-NE group followed the CLX-SLN group (the 24th h: 14 ± 1%, p < 0.0001; the 48th h: 64 ± 0%, p < 0.01). The lowest increase was detected in the CLX-NLC group (the 24th h: 13 ± 1%, p < 0.0001; the 48th h: 66 ± 2%, p < 0.05). The CLX group led to the lowest increase among all groups (the 24th h: 5 ± 0%, p < 0.05; the 48th h: 7 ± 1%, p < 0.05).

3.7. Effects of different CLX applications on HL-60 cells 3.7.1. Cell proliferation index All drug applications decreased cell numbers in comparison to the control group for 48 h (the 24th h: 906333 ± 11,695, the 48th h: 1463000 ± 31,798). CLX-SLN led to the highest decrease among formulations [(the 24th h: 584500 ± 3041, the 48th h: 163333 ± 26,168; p < 0.0001), (36 ± 1% and 89 ± 0% decrease, respectively)]. The mild decrease was detected at the CLX-NLC group [(the 24th h: 770500 ± 1041, p < 0.05; the 48th h: 416667 ± 1193, p < 0.001), (15 ± 1% and 72 ± 2% decrease, respectively)]. The CLX-NE group induced the lowest decrease among the formulations [(the 24th h: 788333 ± 6360, p < 0.05; the 48th h: 442702 ± 2333, p < 0.001), (13 ± 1% and 70 ± 2% decrease, respectively)]. The

3.7.4. Apoptotic and anti-apoptotic protein levels Alterations on apoptotic and anti-apoptotic protein levels done by all applications were clearly seen in Fig. 8C. Effects of formulations were compared with the pure CLX. CLX induced the highest increase in COX-2, MK, Bcl-2, ABCG2 and P-gp levels, p-AKT/AKT ratio in comparison to formulations, however the lowest increase in caspase-3 and Fig. 5. d(0.1), d(0.5) and d(0.9) of placebo and drug loaded formulations during 6 months of storage. (Laser Diffraction (LD) data: volume distribution, PD: production date) (* and ** indicate statistically significant differences in LD data between PD and after 6 months of storage at various temperatures, p < 0.05 and p < 0.01, respectively).

7

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pore size of epithelial cells surrounding tumor vessels expand to be 200 nm-1.2 μm wide, transfer of colloids into tumor cells gets easy [30]. Particle and droplet size measurements also demonstrated physical stability of formulations during the storage period. Nanoparticles maintained their particle size at RT and 40 ± 2 °C while an increase in particle size was observed at 4 ± 2 °C indicating the agglomeration. However, CLX-NE was stable under all thermal conditions. Slight changes in this formulation might be attributed to Ostwald ripening of droplets [31]. Drug peaks were detected in DSC thermograms of CLX-SLN and CLX-NLC in consequence of the presence of drug crystals in the nanoparticle structure (Fig. 4). The peak area of the drug in CLX-SLN was higher (9.95 J/g) compared to CLX-NLC (5.14 J/g). Immediate cooling of the hot NE was accomplished to lead to the recrystallization of the drug prior to the solid lipid in the production process of CLX-SLN. The drug was loaded into nanoparticles with the drug-enriched core drug entrapment model of SLN as reported earlier [7,8]. This drug entrapment model is obtained when the drug concentration is relatively close to or at its saturation solubility in the melted lipid. The drug-enriched core drug entrapment model of SLN provides prolonged drug release since drug is immobilized by the lipid shell surrounding the core (Fig. 7). In the case of CLX-NLC, drug crystals were affirmed to settle in the lipid structure of nanoparticles in a less ordered crystal/amorphous state, i.e. a less pronounced crystalline structure of nanoparticles [32]. Changes in the peak area of the solid lipid were caused by the surface active agent and incorporation of the liquid lipid to formulations independent of any chemical interactions [26]. DSC experiments also showed us that the polymorphic transition of the solid lipid in CLX-SLN and CLX-NLC was accelerated at RT and 4 ± 2 °C in the dark during 6 months of storage (Table 1). CI values displayed changes varying to αpolymorph of the solid lipid in nanoparticles at those temperatures. However, 40 ± 2 °C displayed lack of polymorphic transition. The effect of the drug mobility in the carrier on the diffusion rate became much more obvious when CLX-NE was taken into consideration. Drug mobility within droplets caused a strict biphasic shape of its release profile. While drug molecules close to the outer shell of droplets were being released at first, inner drug molecules were moving to the surface of droplets and then they were released. The cumulative amount of CLX-NE released was only 45.3% within 1 h, demonstrating that there was no burst effect for this formulation. A hydrophobic interaction between the drug and oil may help retain the drug in the droplet protecting NE from the burst effect. Furthermore, PEG chains protected the formulation from the burst drug release [11]. In vitro drug release experiments gave results consistent with DSC findings. CLX-SLN and CLX-NLC displayed sustained drug release profiles inducing drug entrapment models [33]. Drug accommodation in the core and in the less ordered crystal/amorphous structure of nanoparticles contributed the prolonged drug delivery avoiding the burst effect. Those formulations conducted drug release according to the nonFickian anomalous transport pattern (diffusion/lipid matrix erosion). First, less amount of CLX was released near the surface of nanoparticles released during about 4 h and then the drug delivery from the matrix structure took place. This can also be attributed to the perfect sink condition of the release media [34].

Fig. 7. In vitro release profiles of formulations (CLX-SLN, CLX-NLC and CLXNE) and CLX solution in water:propyleneglycol:ethanol mixture (50:25:25) as the release medium.

bax levels was detected in this group. The highest increase in caspase-3 and bax levels was detected in the CLX-SLN group (p < 0.00001, p < 00001) and the lowest increase was induced by the CLX-NLC group (p < 0.0001 for caspase 3, p < 0.01 for bax). The CLX-NLC group led to the highest decrease in COX-2 levels (p < 0.05), the lowest decrease was detected in the CLX-SLN group (p < 0.05). The CLX-NE group led to the highest decrease in Bcl-2 (p < 0.01) and MK levels. The lowest decrease at these levels was detected at the CLX-SLN group (p < 0.05 for bcl-2, p < 0.01 for MK). The p-AKT/AKT ratio was decreased efficiently by CLX-NLC (p < 0.001), CLX-NE (p < 0.01) and CLX-SLN (p < 0.01), respectively. The CLX-SLN group induced the highest decrease in ABCG2 (p < 0.00001) and P-gp (p < 0.00001) levels and the lowest decrease was detected at the CLXNE group (p < 0.001 for ABCG2, p < 0.01 for P-gp). 4. Discussion Spheroidal nanoparticles were obtained with high drug payload. Drug payload of lipid nanoparticles increased with addition of the liquid lipid to the lipid phase (p < 0.05). This can be attributed to the formation of a more suitable medium for the drug to settle into the lipid structure of nanoparticles [26–28]. Drug content of all formulations (CLX-SLN, CLX-NLC and CLX-NE) changed at 4 ± 2 °C during 6 months of storage and additionally at 40 ± 2 °C for CLX-SLN (p < 0.05) (Fig. 3). The highest drug expulsion at 4 ± 2 °C can be attributed to an increase in the polymorphic transition rate of the solid lipid [7,28]. Liquid lipid addition was confirmed to alter particle size insignificantly in general in this study. High drug payload and small particle/ droplet size of SLN, NLC and NE below 150 nm, which are desirable for cellular uptake by cancer cells, contribute great benefits required from sophisticated colloidal drug delivery systems. Decrease in the size of nanoparticles results in easier cellular uptake [29]. Moreover, since

Table 3 Kinetic modelling of drug release profiles of CLX-SLN, CLX-NLC and CLX-NE through dialysis membrane. Formulations

CLX-SLN CLX-NLC CLX-NE CLX solution

Zero order

First order

Higuchi

Korsmeyer-Peppas

r

k0

r

k1

r

kH

r

n

0.9924 0.9337 0.6934 0.6742

0.83 1.34 1.22 65.12

0.7867 0.6442 0.5989 0.8100

0.03 0.02 0.01 1.27

0.9930 0.9909 0.8347 n/a

6.22 10.69 11.04 n/a

0.9940 0.9351 0.9927 n/a

0.87 0.74 0.44 n/a

r: correlation coefficient. Constants of kinetic models: k0, mg%/h; k1, h−1; kH, mg%/h1/2. n: release exponent of Korsmeyer-Peppas model. 8

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Fig. 8. A. Panels of flow cytometric analysis of the apoptotic index. According to the kit's instruction manual, quadrants of panels were defined as: the lower left quadrant of density plots for numbers of viable cells (annexin V−; PI−), the lower right quadrant for numbers of earlier stages of apoptotic cells (annexin V+; PI−), the upper right quadrant for numbers of late stages of apoptotic cells (annexin V+; PI+), and the upper left quadrant for numbers of dead cells. Apoptotic index was calculated as the sum of early and late stages of apoptosis. B and C show the graph of alterations in apoptotic and non-apoptotic protein levels for MCF-7 and HL-60 cell lines at the 48th h, respectively. Data was shown means ± SEM. Mean values were represented in % and calculated as the formula: (the protein levels in drug groups/the protein levels in control group) x 100.

CLX-SLN induced the highest decrease in numbers of MCF-7 and HL60 cells via triggering the apoptosis through the activation of caspase-3, increase of bax levels and the inhibition of bcl-2, p-akt, COX-2, MK and drug efflux protein levels in comparison to all experimental groups as well as the control group. CLX-SLN seems to act in leukemia cells much more effective than breast cancer cells. In addition to this data, CLXSLN seems to facilitate cells' drug intake via the efficient inhibition of drug efflux proteins. Therefore, the drug concentration inside the cell increased and then the drug could act more efficiently. Interestingly, controversial reports can be found for the effect of CLX on these proteins [35,36]. One of them is previous report by Kalalinia et al. [35]. They showed that CLX increased the sensitivity to other drugs such as mitoxantrone, however it increased the expression of ABCG2 in MCF-7 cells. In the same study, they proposed a possible COX-2 independent mechanism for the stimulatory effect of CLX on ABCG-2. Xia et al. determined that CLX at a concentration of 50 μM enhanced the sensitivity of other anticancer drugs as tamoxifen in MCF-7 cells through the inhibition of P-gp, however this effect was independent from COX-2 inhibition [36]. Results of the study by Dharmapuri et al. detected that the overexpression of COX-2 induced the upregulation of Multidrug Resistance Proteins (MRPs) in imatinib resistant (R)-K562 cells which were chronic myeloid leukemia (CML) cells and CLX down-regulated the ABC transporters through Wnt and mitogen-activated protein kinase kinase (MEK) signaling pathways [37]. They concluded that the

combination of CLX with imatinib may be a promising treatment model for overcoming the drug resistance. No study about AML experiments searching for the CLX effect on ABCG proteins was found. All studies were done with CML cells. In concomitant to the study of Dharmapuri et al. [37], but in contrast to studies by Xia et al. [36] and Kalalinia et al. [35], our data showed the inhibitory effect of CLX on two of these drug efflux proteins and may indicate a possible COX-2 dependent mechanism for the inhibitory effect of CLX on ABCG2. The lowest/ highest COX-2 levels with the lowest/highest drug efflux protein levels detected in the present study may show that the activation or inhibition of drug efflux proteins for breast cancer may be done directly through the COX-2 activation or inhibition, however an indirect effect of COX-2 can be found for leukemia. MK, a growth factor with cytokine actions, has several significant biological activities cell growth, cell proliferation, reproduction and tissue repair. MK levels are very high at the embryogenesis stage, it decreases or falls to almost zero in adult tissues [38]. High MK levels were detected in several cancer types including leukemia and breast cancer [39,40]. Previous reports have suggested that the MK gene may be involved in the multidrug resistance [40]. Effects of MK on these proteins were changed due to type of the drug or the drug combination, the concentration of drugs, cell type [25,41–43]. No study was found about CLXs' effect on MK. In our study, we showed for the first time that MK can be a resistance factor for CLX in addition to other resistance 9

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factors, pure CLX also inhibits MK mildly and CLX-SLN can overcome the resistance via the highest effective inhibition of MK for two different type cell lines. According to our data, the inhibition of ABCG2 and P-gp activities may be related to the inhibition of MK indirectly. Phosphorylated Akt is a highlighted molecular target because it contributes to the development of breast cancer and induces resistance to conventional therapies. Arunasree et al. detected that CLX induced apoptosis via inhibiting COX-2 and down-regulating multidrug resistance (MDR)-1expression through p-Akt/Akt/ signaling pathway at IR-K562 cells [44]. Kucab et al. determined that P-Akt was moderately to highly expressed in 58% of primary breast cancers (221/390 cases) and breast cancer cell lines including MCF-7 [45]. They detected that CLX analogues inhibited cell proliferation of several breast cancer cell lines including MCF-7 via p-AKT inhibition. Wang et al. showed that CLX induced p-Akt inactivation through the regulation of Akt signaling independently of its inhibition of NF-κB transcriptional activity in breast cancer cell lines [46]. In the present study, CLX-SLN induced the highest p-AKT activation, but pure CLX induced the highest p-AKT levels among all groups. Consequently, it can be presumed that the inhibition of drug efflux proteins by CLX-SLN may indirectly proceeded through p-AKT/AKT pathway. In addition, Xu et al. showed that MK gene (MDK) led to gastric cancer cell survival and growth via triggering both the Akt and ERK1/2 pathways and upregulates the expression of several cell-cycle-related proteins [47]. Promotion of the growth of mouse embryonic stem cells (mESCs) by MK was shown by Yao et al. [48]. In their study, they found that MK inhibits apoptosis and facilitated the progression toward the S phase, and enhances mESC selfrenewal through PI3K/Akt signaling pathway. In addition, when MK and p-Akt levels for all applications were evaluated for the present study, it might be concluded that MK activated the p-AKT expression and this activation may affect the expression of drug efflux protein levels indirectly.

[This work was supported by the Research Fund of Istanbul University (Project number: UDP-46266)]. Two parts of this study were orally presented in The 3rd International BAU-Drug Design Congress-Novel Methods and Emerging Targets in Drug Discovery and Patented Drug Development in Istanbul, Turkey, 1st-3rd of October 2015 and in The FEBS Congress 2017 in Israel, Jerusalem,10-14th September 2017. The patent application was also done for this project. We thank Yildiz Technical University, Science and Technology Application and Research Center and Assoc. Prof. Dr. Osman Erol at Istanbul University, Faculty of Science, Department of Biology Particle in Istanbul for their valuable contributions in PCS and SEM analysis, respectively. References [1] G. Landskron, M. De la Fuente, P. Thuwajit, C. Thuwajit, M.A. Hermoso, Chronic inflammation and cytokines in the tumor microenvironment, J Immunol Res 2014 (2014) 149185(19 pages). [2] Y. Takada, A. Bhardwaj, P. Potdar, B.B. 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5. Conclusion Physically stable PEGylated nanoformulations (CLX-SLN, CLX-NLC and CLX-NE) of CLX were prepared with high drug payload using Precirol® ATO 5 and Miglyol® 812 as solid and liquid lipids. Cremophor® EL was the most suitable surfactant to provide colloidal carriers of CLX with those lipids, having good physical stability. Cremophor® EL was also the essential excipient in the dispersions for avoiding the phagocytic uptake to modulate biodistribution parameters of formulations and improving their presence in the blood circulation for systemic use. As an addition, particle and droplet size below 200 nm is suitable for colloid transport across the tumor epithelium in general and then for easier cellular uptake. Thus, it was concluded that lipid nanoformulations of CLX optimized in this study contributed advantages of controlled drug delivery in order to provide efficient therapy. The action mechanism of CLX as well as our new designed nanoformulations was also investigated and discussed through MK pathway combined with other well-known pathways (drug-efflux proteins, COX-2) for the first time. SLN, NLC and NE of CLX prepared in this study can be used effectively alone and in combination therapies without the drug efflux protein concern for breast cancer and acute promyelocytic leukemia. We presume that it would decrease side effects of single or combined chemotherapy modalities done with CLX and this would result in good prognosis with increase in the life quality of patients. Acknowledgements This study was supported by TUBITAK (The Scientific and Technological Research Council of Turkey). Project number: 110S435. A part of this study were presented as a poster in Skin Forum 14th Annual Meeting, Percutaneous penetration-measurement, modulation and modelling in Prague, Czech Republic, 4th-5th of September 2014 10

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