Preparation and the Influencing Factors of Timozolomide Liposomes

Abstract:

To prepare timozolomide liposomes for administration through nasal mucous membrane, we studied the factors of the preparation of the liposomes. The timozolomide liposomes were prepared by the ammonium sulphate gradient method; electroscopy and laser particle analyzer were utilized to determine the conformation, size and distribution of timozolomide liposomes; high performance liquid chromatography (HPLC) was applied to determine the entrapping efficiency of timozolomide liposomes; then we studied the influences of the concentration of ammonium sulphate solution, temperature, and the drug-to-lipid ratio on the entrapping efficiency. The average size of timozolomide liposomes was 185 nm; the entrapping efficiency was 90.3%. The entrapping efficiency was enhanced with the increasing of the concentration of ammonium sulphate solution and the rising of temperature, and decreased with the increasing of the drug-to-lipid ratio. The timozolomide liposomes with high entrapping efficiency, small and even particle sizes could be prepared by the simple and convenient ammonium sulphate gradient method. The primary influencing factors on the entrapping efficiency of timozolomide liposomes were the concentration of ammonium sulphate solution, the temperature, and the drug-to-lipid ratio.

Keywords::

• timozolomide
• liposomes
• ammonium sulphate gradient
• HPLC

INTRODUCTION

Timozolomide is a new anti-tumor drug treating neuroglioma and melanocarcinoma, a product of Schering-Plough Corporation. Its main side effects include nasusea, vomiting, asthenia, constipation, and mild arrest of bone marrow. Timozolomide could prolong the survival time of patients suffering from neuroglioma and melanocarcinoma longer than procarbazine. Therefore timozolomide is the relative best anti-tumor drug treating for neuroglioma and melanocarcinoma at the present time [1].

There are many advantages for liposome administration through nasal mucosa; for example, to lower the drug's toxicity and irritation to nasal mucosa, to protect the drug from enzymolisis on the nasal mucosa, to keep a higher drug concentration in administration position, to maintain a sustained release of enveloped drug, and the fact that it has bio-conglutination (especial positive charge liposome) to protect the drug from clearing by nasal cilium [2–4].

To prepare timozolomide liposomes for administration through the nasal mucous membrane, timozolomide could pass the thin basis cranii to enter into the brain along the olfactory nerve cells existing in regio olfactoria. Thus part of timozolomide could round the blood-brain-barrier to enter into the brain directly, decreasing the content of timozolomide in peripheral tissue and lowering the side effects [5].

Timozolomide is a kalescent drug, tending to combine with sulfate ion to form lower soluble sulphate. Liposomes prepared through the ammonium sulphate gradient method seldom leak due to the change of surrounding pH, so we prepared the timozolomide liposomes through the ammonium sulphate gradient method, hoping to obtain stabler liposomes with higher entrapping efficiency [6].

MATERIALS

Timozolomide, Huanrui Pharmaceutical Corporation; egg phosphatidyl choline, EPC, for injection, Sigma; cholesterol, CH, for medicine, Sigma.

Rotatory flash film concentrator, thermostat water bath, CQ-250 ultrasonic cleaner, Agilent HPLC, Beckman laser particle-size analyser, Hitachi JA10T transmission electron microscope.

METHODS AND RESULTS

1. Preparation of Timozolomide Liposomes

We mixed 5g EPC with 1g CH, dissolved with 30ml absolute ethyl alcohol. Then we poured the ethyl alcohol solution into 60ml 0.12mol/L ammonium sulphate aqueous solution (60°C) slowly. We stirred the mixture in 60°C water bath for 2 hours to volatilize the ethyl alcohol, then vibrated it in the ultrasonic cleaner for 10 minutes to obtain the liposomes without drug.

We put the above-mentioned liposomes suspension into a dialytic bag and sealed the bag mouth. The dialytic bag was put into the beaker with 1000ml N.S, dialyzed 3 times at room temperature, 2 hours at a time. Then we put the timozolomide dilute hydrochloric acid solution (1.0g→40ml) into the liposomes suspension, stirred in the ultrasonic cleaner for 15 minutes, put the beaker into the 37°C water bath for 30 minutes, and obtained the timozolomide liposomes.

2. Determination of Shape, Particle Sizes and Distribution of Timozolomide Liposomes

We diluted the right amount of timozolomide liposomes with 500ml water, and determined the particle sizes and distribution by laser particle-size analyzer; we stained the timozolomide liposomes with phosphomolybdic acid and observed the shape with a transmission electron microscope.

The average particle size of timozolomide liposomes was 185nm. Figure 1 shows the distribution and range of timozolomide liposomes size. Figure 2 shows the SEM pictures of timozolomide liposomes (×200,000), seven liposomes sizes, and distinct double layer coating structures.

Figure 1. Distribution and range of timozolomide liposomes sizes.

Figure 2. SEM pictures of timozolomide liposomes (×200,000).

3. Determination of Entrapping Efficiency of Timozolomide Liposomes

3.1 HPLC conditions Stainless steel C₁₈ 5μm chromatography column, 250mm×4.6mm; mobile phase: pH2.7 phosphate buffer/methanol (95/5); flow speed: 1.0ml/min; detection wavelength: 283nm; injection volume: 20μl.

3.2 Chromatography standard curve of timozolomide We weighed 1.0g timozolomide precisely, dissolved it with 50ml dilute hydrochloric acid, then diluted the timozolomide solution with mixed solvent (HCl/ethanol/water, 1/39/60) to prepare a standard solution whose concentrations are 0.2, 0.4, 1.0, 1.4, 1.6, and 2.0 mg/ml; we could get the linear relation from peak area(Y) and concentration(X):

Y = 208.98X +0.42 r=0.9999

3.3 Recovery test of the determination method We weighed 1.0g timozolomide precisely, dissolved it with 50ml dilute hydrochloric acid, mixed 5ml liposomes suspension without drug into 0.5, 2.5, and 5ml timozolomide solution, respectively, dissolved it with mixed solvent (HCl / ethanol / water, 1/39/60), and filtrated with millipore filter. We determined the content of timozolomide by the above-mentioned HPLC method; the mean recovery ratio was 97.64%±4.9% (n=3).

3.4 Determination of entrapping efficiency We added 1.0ml timozolomide liposomes into the top of the SephadexG50 column (1.0cm×10cm), which had been balanced with N.S, eluted with N.S, flow speed was 0.5ml/min, collected 0.5ml eluent in sequence, and detected the eluent through silica gel G thin layer chromatography. The developing agent was normal butanol / glacial acetic acid / water (2:1;1), the visualization reagent was fresh isopyknic mixture of 10% iron chloride solution and 5% red potassium prussiate solution.

Timozolomide liposomes were eluted between 2.5ml and 4.5ml; we collected the liposomes solution, and dissolved it with mixed solvent (HCl / ethanol / water, 1/39/60) to 10ml, determined the content of timozolomide (E) by HPLC. 1.0ml timozolomide liposomes (without elution) sample was dissolved with mixed solvent (HCl / ethanol / water, 1/39/60) to 10ml as the total timozolomide content (T), 1.0ml liposomes (without drug) was dissolved with mixed solvent (HCl / ethanol / water, 1/39/60) to 10ml as the blank control. We determined the timozolomide contents of the 3 solutions; the entrapping efficiency of timozolomide liposomes was the percentage ratio of E to T (90.3); timozolomide carrying in liposomes was 15%.

DISCUSSION

In the process of our experiments, we found that higher entrapping efficiency of timozolomide liposomes arose with higher concentration of ammonium sulphate solution. The higher concentration of ammonium sulphate solution resulted in the higher gradient of ammonium sulphate between the internal and external water phases, the higher pH gradient and driving force, and the higher contents of timozolomide entering the internal water phase.

The temperature of the ammonium sulphate solution is important. In some temperature areas with constant other conditions, higher entrapping efficiency comes with higher temperature of ammonium sulphate solution. At the point of 37°C, timozolomide liposomes were stabler as well as higher entrapping efficiency.

Furthermore, the ratio of drug to liposome is the important influencing factor on the entrapping efficiency of timozolomide liposomes. The higher drug-to-lipid ratio will lead to the higher content of drug gathering in the internal water phase; more H⁺ are neutralized and the gradient of ammonium sulphate and pH will disappear faster, so it is difficult for timozolomide to further enter the internal water phase. In the process of our experiments, when the drug-to-lipid ratio was 1:2, 1:4, and 1:6, the entrapping efficiency of timozolomide liposomes was 28%, 62%, and 90%, respectively. The results suggest that the higher drug-to-lipid ratio led to the lower entrapping efficiency.

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

This paper was first published online as an Early Online article on 10 November 2009.