1. INTRODUCTION:

Cancers of the digestive system are the third leading cause of death from cancer worldwide [1]. According to statistics, resection is effective for not more than 50% of patients, but for other patients it is necessary to pass the course of chemotherapy. Application of modern chemotherapeutic agents allows achieving good results in the fight against disease [2]. However, this class substances has no properties of selectivity between normal and pathologically transformed tissue.

Oxaliplatin is a promising medicine, complex compound, the representative of the third generation platinum drugs. Central atom of platinum in the oxaliplatin molecule is surrounded by two ligands - oxalate and diaminocyclohexane, located in trans state[3]. Oxaliplatin, as other platinum cytostatic, interacts with cell DNA to form both inter- and intra-strand cross links thus blocking replication. The inability of DNA replication and transcription leads to the inhibition of protein synthesis causing cell death[4,5].

Oxaliplatin is used in both monotherapy and in combination with other cytostatics. Oxaliplatin is effective in treatment of tumors having resistance to cisplatin and carboplatin[6].

Despite the advantages of oxaliplatin in the therapy, it is, like most cytostatic has high level of toxicity, which limits its clinical use. Especially should be noted neutropenia and extravasation. Creating of oxaliplatin liposomal form give potential to reduce mentioned toxic effects during application and will give the possibility to increase the dose upon administration. Recovering of damaged cell's membranes by phospholipids and «EPR effect» lead to toxicity reducing and increase the accumulation of liposomal nanoparticles in the tumor[7]. This can be used for improving the quality of life for patients, and to improve treatment efficiency by increasing of therapeutic dose also[8]. We are working under development of liposomal oxaliplatin for use in the chemotherapy of tumors. In present work, method of preparing liposomes by ion sorption oxaliplatin molecules on the lipid membrane, followed by homogenization under high pressure was used.

The structure of oxaliplatin molecule is shown in Fig. 1. Electron density in the molecule is redistributed with such effect that oxygen atoms are connected to the platinum atom, carry a negative charge, and the platinum atoms in turn, are positively charged. Such structure provides possibility for oxaliplatin to create complexes both with positively and negatively charged molecule.

Fig. 1. Structure of oxaliplatin molecule.

Purpose:

The purpose of this work is the development and standardization of technological parameters of extrusion in the preparation of liposomal form of oxaliplatin, definition of the process parameters and characteristics of final product.

MATERIALS AND METHODS:

For liposomes preparation egg phosphatidylcholine by Lipoid, Germany, was used. Cholesterol was purchased from Sigma-Aldrich, USA. Dipalmitoylphosphatidyl glycerol was purchased from Avanti Polar Lipids, USA. The lipid film was obtained by evaporation method, with rotary evaporator Buchi Rotovap R-210, Switzerland, equipped with a vacuum controller, at a residual pressure of 15 mbar. For homogenization, an ultrasonic bath "Sapphire" Russia, with volume 2.8 liters, at operating frequency of 35 kHz, equipped with 130 W power generator, and extruder Microfluidics Microfluidizer M-110P, USA was used. Liposome size and the zeta potential was study by laser diffraction method on Zetasizer Nano ZS, Malvern Instruments, United Kingdom. Determination of encapsulation degree was carried out by HPLC gel filtration sistem, with Shimadzu LC-20instrument, Japan, with the following conditions:

- Column - GE Healthcare Tricorn Columns 150 x 5 mm;

- Mobile phase - 0.01 M NaH₂PO₄, pH 5.0;

- Mobile phase flow rate - 1 mL / min;

- The volume of the sample - 1 ul without dilution;

- Detection - spectrophotometry at a wavelength of 220 nm. Determination was performed based on the previously developed technique [9].

RESULTS:

In therapeutic use of liposomes, may be observed decreases of blood concentration of free oxaliplatin, also decreasing its contact with the blood vessels at the injection site, which reduces the effect of vascular irritation during administration. Particla size has a great influence to therapeutic efficiency of liposomes. The size of some of the most widespread liposomal cytostatic, approved by FDA - Myocet, ( Elan Pharma, USA), is in the range 120 - 150 nm, and «Doxil», (Janssen Products, USA), is in the range 80 - 100 [10,11]. Consequently, the size of the liposomes, successfully used in clinical use, exist within range of 80 - 150 nm.

At the stage of pharmaceutical development for liposomal drug products, we can use the method of "funnel quality", or "quality gate", and at the homogenization stage to reduce desirable range to 90 - 120 nm, with the expectation that at the stage of qualities control we will get guaranteed parameters of nanoparticles size.

In the process of liposomal oxaliplatin pharmaceutical development we have found that the best membrane lipid composition is: egg phosphatidylcholine / cholesterol / dipalmitoylphosphatidyl glycerol in a ratio of 50/20/30 % by weight. Obtained lipid film was hydrated with a solution of oxaliplatin with a concentration of 4 mg / ml. With such value of oxaliplatin composition and concentration, encapsulation degree was reached at least 60% for liposomes in step prior to freeze drying. Technology for liposomes preparation consisted of the following: the lipid film prepared by dissolving the mentioned above lipids in chloroform. To achieve completeness of dissolution for dipalmitoylphosphatidyl glycerol was used treatment in an ultrasonic bath until the disappearance of opalescence. The solvent was removed with a rotary evaporator. The process of dissolution and evaporation was carried out at a temperature not exceeding 30 °C. Next, it was carried out lipid film hydration by oxaliplatin solution with concentration of 4 mg / ml, was applied stirring on a platform at a speed of 100 rev / min in a round bottom flask for one hour. The resulting "coarse" emulsion is briefly treated in an ultrasonic bath, to remove visible particles. Stages of hydration and preparation a coarse emulsion were carried out at a temperature not higher than 20°C. Resulting emulsion was homogenized in an extruder Microfluidizer M-110P in a mode of 900 atm. By the time of extrusion lipid concentration in the emulsion was 40 mg / ml. To achieve liposomes with size 106 nm was applied 9 extrusion cycles at 20 ° C. In the last stage of oxaliplatin liposomes preparation cryoprotectant solution was added to the emulsion and it was diluted with water for injection until the oxaliplatin concentration of 2 mg / ml and the total lipid concentration of 20 mg / ml. Resulting emulsion was filtered through 0.22 mkm polyethersulfone (PES) membrane, in aseptic condition. The particle size at this point was 103 nm, along with 66% of encapsulation degree. The zeta potential of the liposomes was measured as "- 68.3" mV. The resulting emulsion was aggregatively stable in the period of observation - 5 hours and showed no signs of sedimentation.

Extrusion was a major step, which determines the dimensional properties of the final product. Extruder mode at 900 atm allows to smoothly adjust liposomes dimensions during the process of pharmaceutical development, but at the same time, during the industrial process, this mode may be inefficient from point of view of labor costs, and time of equipment use. The extruder allows to perform operation up to 2000 bar. We studied the possibility of increasing the pressure during extrusion process. We invistigated extrusion modes with pressure of 900 atm 1,100 atm, 1300 atm, 1,500 atm with obligatory cooling to 20 °C. The emulsion was prepared as described above. The criterion of development was the achievement of size 90 - 120 nm with the lowest number of cycles. A plot of the particle size from applied pressure and the number of extrusion cycles are shown in Fig. 2.

Fig. 2 shows that the particle size and the number of cycles depends on the pressure during the extrusion. At the same conditions, when at 900 atm is necessary to apply 9 cycles for liposome size 106 nm, at 1500 atm number of cycles is reduced to 4, with the resulting size of 102 nm.

After extrusion process, the crioprotector solution was added to resulting emulsion, and it was diluted with water for injection until the oxaliplatin concentration of 2 mg / ml and the total lipid concentration of 20 mg / ml, as described above. Particle size values, zeta potential and encapsulation degree were determined in the obtained samples. The values given in Table. 1.

Table 1. Values of particle size, zeta potential and the degree of encapsulation, mesured in liposomes.

No.	Extrusion mode	Particle size, nm	Zeta potential, мВ	Encapsulation degree, %. Р=0,05; n=3
1	900	109	-67,9	67±0,72
2	1100	111	-66,4	64±0,84
3	1300	106	-68.6	66±0,77
4	1500	102	-66,3	67±0,81

To confirm the reproducibility of the technology, three laboratory series of liposomal oxaliplatin was made. In the manufacture process was used extrusion mode 1500 atm. In final liposomes were mesured sich characteristics, as: particle size, zeta potential, and encapsulation degree. The results are shown in Table 2.

Table 2. Mesured characteristics of three laboratory series of liposomal oxaliplatin. Extrusion at 1500 atm..

No. of butch	Particle size, nm	Zeta potential, mV	Encapsulation degree, %. Р=0,05; n=3
1	103	-66,8	65±0,75
2	104	-65,4	67±0,79
3	98	-66,3	64±0,71

RESULTS AND DISCUSSION:

Extrusion at 1500 atm , as seen in Figure 1 allow to reduces the number of cycles, in comparison with 900 atm mode, more than twice. At the same time, liposome size is within the desireble specified range of 90 - 120 nm. Magnification of pressure take place without loss of encapsulation degree, which confirm the possibility to use the mode in a scale of industrial technology. Also, during the obtained samples examination, value of the zeta potential was determined at the same level, from "-65" to - "-66" mV, what was an indication of high stability of the liposomal emulsion. Production of three consecutive laboratory series of liposomal oxaliplatin showed reproducibility characteristics - size, zeta potential and the degree of encapsulation for the active substance. It is shown that extrusion at 1500 atm. and at 20°C lead to formation of standard liposomes in the size range 90 - 120 nm with reproducible encapsulating.. From Table 2 can be seen that the characteristics of the three obtained series of the product is practically identical, that indicating the stability of the developed process.

CONCLUSIONS:

As a result of the work was developed the optimal extrusion technology, a main process in the production of liposomal formulations. The most optimal from point of view of interrelation "quality of the product - time production" was shown extrusion mode at 1500 atm. Researched mode was standardized by working parameters. Was performed characterization of final products by following parameters: particle size, zeta potential, encapsulation degree. The results will be used in the manufacture of liposomal form of oxaliplatin for use in oncology chemotherapy.

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Received on 28.11.2016 Modified on 26.12.2016

Research J. Pharm. and Tech. 2017; 10(3): 785-788.

DOI: 10.5958/0974-360X.2017.00147.0