Optimisation of processing variables effective on self ...

IET Nanobiotechnology Research Article

Optimisation of processing variables effective on self-assembly of folate targeted Synpronic-based micelles for docetaxel delivery in melanoma cells

ISSN 1751-8741 Received on 16th December 2014 Revised on 18th March 2015 Accepted on 24th April 2015 doi: 10.1049/iet-nbt.2014.0076 www.ietdl.org

Somayeh Taymouri 1, Jaleh Varshosaz 1 ✉, Farshid Hassanzadeh 2, Shaghayegh Haghjooy Javanmard 3, Nasim Dana 3 1

Department of Pharmaceutics, School of Pharmacy and Novel Drug Delivery Systems Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran 2 Department of Medicinal Chemistry, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran 3 Physiology Research Center, Isfahan University of medical sciences, Isfahan, Iran ✉ E-mail: [email protected]

Abstract: Polymeric micelles (PMs) were formulated as nano carriers for docetaxel intended for both intravenous administration and improve therapeutic efficacy of the drug. The PMs were formulated using folic acid conjugated Synpronic F127-cholesterol copolymer and were optimised using a 23 full factorial design. The effects of different formulation variables were evaluated on the particle size, entrapment efficiency (EE), zeta potential and release efficiency of the micelles. The in vitro cytotoxicity of DTX-loaded FA targeted micelles was studied on B16F10 melanoma cells which over expressed FA receptor. Among the studied single factors, solvent type was the most effective parameter on the EE and release efficiency. Polymer/drug ratio had the most considerable effect on the particle size while, zeta potential was more affected by temperature. Finally, the PMs with polymer/drug ratio of 12 prepared at 25°C by dimethyl sulfoxide as the dialyzing solvent was shown to be the optimum formulation with desirability factor of 84.9%. The optimised formulation exhibited a particle size of 171.3 nm, 99.59% drug EE, zeta potential of −7.80 mV, drug release efficiency of about 70% at 144 h and polydispersity index of 0.32. The MTT assay indicated DTX-loaded FA targeted micelles were significantly more cytotoxic than non-targeted micelles and free drug.

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Introduction

Cancer is a life threatening disease with an annually increasing incidence. About 1 660 290 new cases were diagnosed with cancer and 580 350 died from it (about 1600 people per day) in the USA in 2013 [1]. In 2007, cancer was the reason for 13% mortalities worldwide [2]. Cancer is caused by uncontrolled proliferation of abnormal cell. Depending on the type of cancer, cancerous cells can metastasize to other sites of body through blood or lymphatic circulation and influence other parts of body. Current treatment of cancer includes surgery, radiation and chemotherapy [3]. However, the main treatment for localised and metastasized cancer is chemotherapy with cell killing drugs. Cytotoxic drugs have been used either alone or combined with other drugs or other therapies. However, severe toxicity and low therapeutic index of chemotherapeutic agents limit their application in cancer therapy. Drugs used in chemotherapy not only affect cancerous cells, but also influence other proliferating normal cells such as hair follicles, blood cells and gastrointestinal cells [4]. Other factors that limit use of chemotherapeutic agents are their low water solubility and their need for special solvent for formulation which could lead to toxicity [5]. So far, many efforts have been made to develop a new drug delivery system to make a high local concentration of drug in tumour tissue and reduce side effects caused by solvent system and non-specific distribution [6]. Among different drug delivery systems, polymeric micelles (PMs) have gained considerable attention. PMs are core-shell structure of polymeric materials composed of hydrophobic and hydrophilic block monomers that self-assemble in water upon critical micelle concentration (CMC) because of incompatibility of hydrophobic block with surrounding aqueous media and tendency to aggregate via hydrophobic-hydrophobic interaction. Hydrophobic micelle

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core serves as a drug reservoir stabilised by hydrophilic shell. Hydrophilic polymer provides steric hindrance, limits opsonins adsorption and prevents being scavenged by reticuloendothelial system (RES) which contribute to increase in blood circulation time [7]. They provide some advantages in cancer therapy, including: increase in water solubility of poor soluble drugs, improvement of drug bioavailability, reduction in drug degradation and small size which is another factor contributing to prolongation of blood circulation time and increase in tumour accumulation by enhanced permeability and retention effect [8]. For developing a suitable carrier system, selecting proper polymeric material is necessary. Among different amphiphilic materials, Synpronic copolymers have been widely considered in drug delivery. Synpronic copolymers, amphiphilic synthetic polymer that is arranged in the triblock structure (PEO)x-(PPO)y-(PEO)x, works as biological response modifiers, increase sensitivity of multidrug resistance tissue to anticancer drugs because of their potential to inhibit P-glycoprotein and have been widely used in pharmaceutics thanks to their suitable properties such as biocompatibility, low toxicity and weak immunogenicity [9]. Synpronic micelles have also been demonstrated as promising drug carriers to increase drug solubility and bioavailability [10]. However, Synpronic copolymers cannot form stable micelles in water because of high CMC and are susceptible to micelle dissociation upon dilution [10]. Cholesterol is a natural sterol that plays a main role in maintaining cell integrity and also many body activities. It has been used to modify polymers backbone because of highly hydrophobic sterol structure, biocompatibility and the ability of cholesterol bearing materials to self-assemble. Cholesterol bearing materials have also been reported to have the higher drug loading capacity and stability compared with those with long alkyl chain [11, 12]. In addition, cholesteryl hemisuccinate has anticancer

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effect because of its ability to inhibit DNA polymerase and DNA topoisomerase enzymes which contribute to DNA replication, repair and cell division [13]. Conjugation of cholesteryl hemisuccinate to Synpronic copolymer might overcome the instability of Synpronic copolymer. Although nanopolymeric micelles can enhance drug accumulation in tumour tissue by passive targeting, linking of an active targeting agent was suggested to selectively increase drug delivery to tumour tissue and enhance uptake of drug carriers. Among various targeting ligands, folic acid (FA) has attracted much attention as a targeting ligand to cancerous cells over-expressed folate receptor because of its low molecular weight, poorly immunogenic and high binding/ recognition affinity to folate receptors [14]. Docetaxel (DTX), a semi-synthetic analogue of paclitaxel, is an antimitotic taxane drug with demonstrated excellent anticancer effect against melanoma, breast, ovarian, prostate and lung cancer. Parenteral dosage form of DTX is formulated with polysorbate 80 and ethanol because of its low water solubility and marketed as Taxotere®, but its clinical application was hampered because of side effects induced via solvent system or the drug itself through non-specific distribution throughout the body [6, 15]. The purpose of this study is to prepare and optimise the factors that affect physicochemical features of PMs prepared from FA conjugated cholesterol-Synpronic F127 (PF127-Chol) copolymer to develop a suitable carrier for delivery of DTX in a fatal type of cancer, that is, melanoma which is growing with a high rate of incidence. Despite numerous attempts for treating metastatic melanoma, conventional therapies including systemic chemotherapies have not been promising. The most cytotoxic agents have low molecular weight, which leads to rapid excretion, non-specific distribution and poor therapeutic index. Recent developments in nanoparticulate drug delivery systems are able to improve the drug efficacy and safety, and offer more promising approaches in treating melanoma.

solvent and free drug. The medium was changed every 2 h until 8 h. Then, the obtained micelle dispersion was freeze dried for further studies. To evaluate the effect of processing variables on particle size, zeta potential, entrapment efficiency (EE) and release efficiency, the Design Expert software (ver. 7.2, USA) was used. Three different variables, including polymer/drug ratio, organic solvent and dialysis temperature were studied each in two levels. Eight different formulations were designed by a general full factorial design (Table 1). All experiments were done in triplicate. The optimum conditions were determined by an optimisation process to yield a heightened performance. 2.3

Scanning electron microscopy (SEM)

The morphology of DTX-loaded PMs was evaluated using electron microscopy. A small amount of freeze dried micelles were coated with gold under vacuum using a sputter coater before SEM (FE-SEM; HITACHI S-4160, Japan). 2.4 Particle size, poly dispersity index (PDI) and zeta potential of the micelles The mean particle size, PDI and zeta potential of DTX-loaded micelles were determined by dynamic laser scattering using Malvern nanosizer (ZEN3600, Malvern Instruments Ltd, UK). Appropriate amount of freeze-dried sample was dispersed in 1 ml of water then poured in quartz cuvette. All measurements were done in triplicate at room temperature and fixed scattering angle of 90°. 2.5

Determination of DTX encapsulation efficiency

Synpronic F127 (PF127), cholesterol (Chol), succinic anhydride, FA, acetonitrile (ACN), dimethyl sulfoxide (DMSO), 1,1′-carbonyldiimidazole, 4-dimethyl amino pyridine, dicyclohexylcarbodiimide, dimethylformamide (DMF) and methanol were obtained from Merck Chemical Company (Germany). DTX was obtained from Cipla (India). Dialysis bag (cut off: 6000 − 8000 Da) was from Sigma Company (USA).

For quantification of DTX loaded in micelles, 1 mg of freeze dried sample was dissolved in appropriate amount of ACN:water (50:50) by sonication. The sample was filtered through 0.45 µm filter and DTX amount was determined using Water HPLC equipped with 515 HPLC pump, dual l absorbance detector and reverse phase (RP) C18 column (Fortis, 250 mm × 4.6 mm, 5 µm). The mobile phase contained the mixture of ACN:water (65:35) at flow rate of 1 ml/min. The calibration curve was recorded in concentration range of 0.250–40 µg/ml in ACN. Injection volume was about 40 µl. The UV detector was set at detection wave length of 230 nm. Drug loading efficiency or EE was determined from the ratio of the determined amount of the drug in 1 mg of the freeze dried sample to the used amount of drug in every mg of the sample.

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2 2.1

Materials and methods Materials

Preparation of DTX-loaded PMs

FA grafted PF127-Chol copolymer was prepared as described in our previous report [16]. DTX-loaded micelles were prepared by dialysis method. For this purpose, a constant amount of DTX and different amounts of copolymer (Table 1) were dissolved in DMSO or DMF by sonication method. For producing PMs, the solution of DTX and the copolymer were dialyzed against distilled water for 24 h using dialysis bag (cut off: 6000–8000) to remove organic Table 1 Studied formulations of FA conjugated cholesterol-Synpronic F127 copolymeric micelles Formulation code P3S25 P12S25 P3F25 P12F25 P3S40 P12S40 P3F40 P12F40

Polymer/drug ratio

Solvent type

Dialysis temperature, °C

3 12 3 12 3 12 3 12

DMSO DMSO DMF DMF DMSO DMSO DMF DMF

25 25 25 25 40 40 40 40

In vitro release of DTX

Release of DTX from micelles was evaluated by dialysis method. Appropriate amount of the freeze-dried sample was dispersed in phosphate buffer solution (PBS) at pH 7.4, placed in dialysis bag (cut off: 6000–8000) and immersed in appropriate amount of PBS at pH 7.4 containing 0.5% Tween 80. At predetermined time intervals, appropriate amount of medium was removed and replaced with fresh medium. The amount of released drug was determined by RP-HPLC as described above. To study the drug release kinetic, DTX release data obtained from different formulations were fitted with various kinetic models including Baker Lonsdale (3/2 × [1-(1-Qt)2/3]-Qt = kBt), Higuchi (Qt = kHt 1/2), Hixson Crowell (Q0 1/3-Qt 1/3 = kst), first order (lnQt = lnQ0 + k1t), zero order (Qt = Q0 + k0t) and Peppas model (Qt/Q0 = ktn) [17]. Where Q0 is initial amount of drug in dosage form, Qt is amount of drug released at time t, t is sampling time, k is release constant and n is release exponent. The best model was selected based on the highest correlation coefficient (r2). To evaluate mechanism of drug release, release exponent (n) was calculated. When n value is equal or less than 0.5, Fickian diffusion is dominant mechanism of drug release. When 0.5 < n < 1, the release mechanism follows non-Fickian (anomalous drug

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Fig. 1 Schematic representation of self-assembling micelles of folate targeted Synpronic/cholesterol

Table 2 Physicochemical properties of DTX-loaded nanoparticles Formulations P3S25 P12S25 P3F25 P12F25 P3S40 P12S40 P3F40 P12F40

Drug loading efficiency, %

Drug loading capacity, %

Particle size, nm

Zeta potential, mV

PDI

Release efficiency, RE144%

65.39 ± 1.05 99.59 ± 2.79 103.18 ± 0.06 52.33 ± 5.74 100.04 ± 2.48 100.07 ± 3.20 83.45 ± 0.70 72.17 ± 1.14

16.34 ± 0.26 7.66 ± 0.21 25.79 ± 0.015 3.45 ± 0.11 25.01 ± 0.62 7.69 ± 0.246 20.86 ± 0.17 5.55 ± 0.087

186.93 ± 17.70 171.30 ± 6.42 260.57 ± 62.97 83.46 ± 13.30 84.04 ± 6.04 89.26 ± 10.63 213.23 ± 6.98 140.80 ± 3.70

−15.60 −7.80 −8.50 −7.60 −5.85 −5.13 −4.72 −4.81

0.36 ± 0.05 0.32 ± 0.07 0.43 ± 0.24 0.78 ± 0.21 0.52 ± 0.09 0.59 ± 0.05 0.38 ± 0.04 0.24 ± 0.03

64 ± 1 70 ± 2 40 ± 1 67 ± 2 78 ± 1 80 ± 4 50 ± 1 46 ± 4

diffusion) model. For n value = 1 drug release follows zero order mechanism. If n value is higher than 1 supper case ІІ release mechanism occurs [17]. 2.7

In vitro cytotoxicity assays

MTT assay was used for comparing the cytotoxicity of free DTX, DTX loaded in non-targeted micelles and DTX loaded in FA targeted micelles. The folate receptor positive cells of B16F10 melanoma were provided from Pasteur institute (Iran). The medium of cells cultivation was RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were incubated in atmosphere containing 5% CO2 at 37°C. Free DTX solution was prepared by dissolving appropriate amount of drug in DMSO 1%. B16F10 cells were seeded in 96 well plates at density of 5 × 103 and incubated for 24 h to allow cell attachment. Then, the cells were exposed with different concentrations of free DTX, DTX loaded in non-targeted micelles and DTX loaded in FA targeted ones for 48 h. At the end of incubation time, 20 µl of MTT solution (5 mg/ml concentration) was added to each well and it was incubated for further 3 h. Then formazan crystals were separated from the medium and dissolved in 150 µl of DMSO in each well. The absorbance of each well was measured at 570 nm using a microplate reader. The medium was used as blank. For analysis the toxicity of the polymeric micelle carriers both non-targeted and targeted micelles were tested as described above in equivalent concentration of material used in drug-loaded micelles. The number of viable cells was determined using the following equation: (see equation at the bottom of the page)

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Results and discussion

Drug-loaded PMs are prepared by a variety of techniques including direct dissolution, solvent evaporation, solid dispersion and dialysis method [18]. In this study, DTX-loaded PMs were prepared with Cell viability% =

Fig. 2 SEM of the optimised formulation of folate conjugated Synpronic F127-cholesterol micelles

dialysis method. DMF and DMSO were selected as solvent because of their high ability to dissolve both drug and copolymer and their miscibility with water [19]. Following solvent exchange, self-assembled micelles are formed by hydrophobic interaction between hydrophobic block leading to their aggregation. Fig. 1 shows schematic representation of the formed micelles. The properties of DTX-loaded micelles including, particle size, PDI, zeta potential, EE and release efficiency are shown in Table 2. The SEM morphology of the micelles exhibited smooth spheres with moderate size distribution (Fig. 2). Fig. 3 shows that the most effective factors on the DTX loading efficiency in the micelles are the interaction of polymer/drug ratio and solvent type. Among the studied single factors, the solvent type was more effective than temperature and the polymer/drug ratio. Molecular volume and concentration of drug, polymer concentration, nature of core and shell forming blocks, physical

Mean absorbance of sample − mean absorbance of blank × 100 Mean absorbance of control − mean absorbance of blank

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Fig. 3 Contribution of different studied parameters and their interactions to DTX loading efficiency Particle size, zeta potential and release efficiency in folate targeted micelles of Synpronic F127-cholesterol

state of micellar core and drug/core compatibility are factors influencing on drug EE [20]. As shown in Fig. 4a, EE increased with increase in polymer/drug ratio which can be explained by accommodation of drug more effectively in greater amount of copolymer [21]. This finding is in agreement with the study of Seo et al. [22]. He showed decrease in the methotrexate encapsulation by increase in drug feeding ratio. Jeong et al. [23] also reported similar results in incorporation of adriamycin in PMs of poly(benzyl-l-glutamate)/poly(ethylene oxide). However, statistical analysis of drug loading efficiency data with Design Expert software showed that when micelles were prepared by DMSO at 40°C (Fig. 4d), EE did not change significantly with polymer concentration because of more effectiveness of temperature on EE (Figs. 3 and 4d). Unexpectedly EE of micelles prepared by DMSO was higher than those prepared from DMF (Fig. 4b) while micelles prepared from DMF were thought to have higher EE because of their larger size. In agreement with our study, the study conducted by Jung et al. [24] also showed that nanospheres of hydrophobised pullulan prepared by acetone had higher EE than those prepared from DMF and DMSO despite their smaller particle size. This result could be attributed to the difference in solubility and miscibility of copolymer, drug and solvent or between water and solvent, which would affect the physicochemical properties of micelles including

size and EE [25]. As the loading temperature increased, the DTX EE increased (Fig. 4c). Wei et al. [26] stated mixed micelles of Synpronics P123 and F127 with certain proportions could form soft gel at high temperature, which caused more effective drug loading Fig. 4e shows interaction effect of solvent on EE in different polymer concentration at constant temperature. The Fig. 4 indicates that EE increased as the polymer concentration increased when nanoparticles prepared from DMSO. However, this effect was reversed when nanoparticles were prepared from DMF. The possible reason may be less miscibility of DMF with water, fewer precipitation rate of copolymer and more opportunity of removing free drug from the dialysis bag during drug encapsulation Statistical analysis with Design Expert software showed that EE in the micelles prepared by DMSO increased as temperature increased at constant polymer/drug ratio; however, it did not significantly change in case of the micelles prepared by DMF (Fig. 4f ). This finding could be because of lower viscosity of DMSO as temperature increased compared with DMF. This caused increase in miscibility with water, facilitated the solvent departure from dialysis bag and increased rate of copolymer precipitation, all of which increased the drug entrapment in nanoparticles. Particle size of nanoparticles is an important parameter that affects longevity in blood circulation and endocytosis by tumour cells. Sufficiently small particles (