6-Thioguanine loaded magnetic microspheres as a new drug delivery system to cancer patients

Magnetic microspheres of 6-thioguanine were prepared by continuous solvent evaporation technique. An attempt was made to target the magnetic microspheres to the cancerous site. Poly lactic acidpolyethylene glycol copolyester (PLA-co-PEG) was used as a polymer. Microspheres were characterized in terms of percentage practical yield, micromeritic properties, particle size, swelling kinetics, magnetic responsiveness, magnetite content and in vitro drug release study. Phosphate buffer pH 7.4 was used for in vitro release study. Microspheres were found to give sustained release pattern. Reticuloendothelial clearance can be minimized and target site specificity can be increased.


INTRODUCTION
6-Thioguanine is a purine antagonist and is a highly effective anticancer drug.In the body, 6-thioguanine is converted to corresponding monoribonucleotides which inhibit the conversion of ionosine monophosphate to adenine and guanine nucleotides.It is highly useful in childhood acute leukemia, choriocarcinoma and solid tumors (Jharap et al., 2011).Mercaptopurine however can be employed as purine antagonist but it causes more nausea and vomiting than 6-thioguanine.Magnetic microspheres, as delivery system, are very much important which localizes the drug to the disease site.In this larger amount the freely circulating drug can be replaced by smaller amount of drug (Tripathi, 2008).The different types of therapeutic magnetic microspheres are: 1.These are used to deliver chemotherapeutic agent to liver tumors.2. Drugs like proteins and peptides can also be targeted through this system.3. Diagnostic microspheres can be used for imaging liver metastases and also can be used to distinguish bowel loops from other abdominal structures by forming nano size particles supramagnetic iron oxides.
Magnetic microspheres have potential use as magnetic seeds for drug delivery.Magnetic fields are believed to be harmless to the biological systems and adaptable to any part of the body.Magnetic microspheres ensure that the maximum amount of dose can be deposited and released in a controlled manner in selected non-RES organs.The range of control over matter allows noninvasive surgery and the ability to pass through tissue and even cell walls instead of lysing them to obtain internal access to the material.The above advantages make the magnetic microspheres an ideal candidate for controlled and targeted drug delivery system.Magnetic microspheres are most stable; also they are recyclable and reusable (Kakar and Singh, 2014).

MATERIALS AND METHODS
6-Thioguanine was obtained from Sigma Aldrich.Poly lactic acidpolyethylene glycol (PLA-co-PEG) copolyester was synthesized from oligomer of L-lactic acid and poly ethylene glycol (PEG) using stannous octoate as catalyst.6-Thioguanine containing poly lactic acid-polyethylene glycol copolyester (PLA-co-PEG) magnetic microspheres were prepared via continuous solvent-evaporation method.The morphologies of prepared magnetic microspheres were evaluated by scanning electron microscopy (SEM).Drug release was observed in phosphate buffer saline (pH 7.4).

Preparation of magnetite
1. Nitrogen gas was flushed through the flask charged with 8.9 g of geoethite, 9.94 g of FeCl 2 .4H 2 O along with 250 ml of distilled water and 50 ml of 2 M sodium hydroxide while stirring vigorously 2. Reaction mixture was heated to reflux.During transformation the pH of the reaction mixture fell to 8. 3. Black colored precipitates were formed.4. Precipitates were filtered and dried at room temperature (Mukherjee, 2012).

Preparation of magnetic microspheres
1. Drug and polymer were dissolved in appropriate volatile organic solvent and then magnetite is added to this solution along with stirring in order to form a homogeneous suspension.2. The suspension was added to an immiscible auxiliary solution along with vigorous stirring.3. Volatile organic solvent was evaporated slowly at 22 to 30°C to form microspheres (Dejagar et al., 2003).4. Microspheres were centrifuged and freeze dried and stored at 4°C (Dhananjay and Nilofar, 2010;Corrigen and Helay 2003).

Determination of percentage yield of microspheres
Thoroughly dried microspheres were collected and weighed accurately.The percentage yield was calculated using formula: Percentage yield = (Practical yield/Theoretical yield) × 100 82.64 was the percentage yield of the microspheres recovered.

Micromeritic properties
Accurately weighed microspheres were poured gently through a glass funnel into a graduated cylinder exactly to 10 ml mark.Initial volume was noted.Bulk density and tapped density were noted using tapping method using 10 ml measuring cylinder (Kakar et al., 2013).Angle of repose (θ), Hausner's ratio (H) and Carr's index (%C) were calculated to study the flow properties of microspheres by using the following formulas: Where, h is height and r is radius of the pile, respectively.
Where, Dt is tapped and Db is bulk density, respectively.Table 1 shows standards for flow properties (as per USP30-NF25 specifications).Figure 1 shows flow properties of prepared magnetic microspheres of 6-Thioguanine.Flow properties of prepared magnetic microspheres of 6-Thioguanine were found to be excellent with Carr's index (9.89%),Hausner ratio (1.11) and angle of repose (29.5).

Particle size
Particle size was determined by SEM.Scanning electron photomicrographs of formulation was used to determine the average particle size of magnetic microspheres of 6-thioguanine.Average particle size was found to be 20 µm.Particles were found to be nearly spherical in shape.Figure 2 shows the SEM image of 6thioguanine loaded microspheres.

Measurement of swelling kinetics of magnetic microspheres
Dried microspheres were immersed in distilled water at different predetermined time.Then the sample was removed from distilled water and was frequently weighed after trapped with a filter paper to remove excess water on the surface.Thus, the wet weight of the microspheres was recorded during the swelling period at regular time intervals (Prasanth et al., 2011).Swelling ratio was found to be more during day 2 and day 3 as compared to day 1. Figure 3 shows the swelling ratio of prepared 6-TG magnetic microspheres.

Magnetic responsiveness of 6-thioguanine loaded microspheres
The apparatus consist of a pump (aerator), which pumped air into the flask containing normal saline (Zhang et al., 2007).Microspheres content of the collected samples were then evaluated using UV-Vis spectrophotometer at 342 nm. Figure 4 shows the apparatus for measuring magnetic responsiveness of 6-TG magnetic microspheres.Prior to injection, microspheres (25 mg/ml) were dispersed in normal saline containing 0.1% w/v Tween 80 and a stock solution was prepared.A flow of 0.5 cm/s of normal saline, resembling the blood flow rate passing through the capillaries, were established.A 1 ml aliquot of the microspheres suspension in the test vehicle was then injected into the injection site.The 8000 G magnetic field was established for 15 min and one sample was collected every minute.The magnetic field was then removed and samples collected for a further 5 min.Microspheres content of the collected samples were then evaluated using UV-Vis spectrophotometer (Shimadzu 1800; Vimal et al., 2007) at 342 nm wavelength.Tables 2 and 3 shows microsphere content (%) retained in presence and absence of magnetic field, respectively.Figures 5 and 6 shows microsphere content (%) retained in presence and absence of magnetic field, respectively.

Determination of magnetite content
Determination of magnetite content in prepared magnetic microspheres was conducted by employing a conventional titrimetric method using thiosulphate and potassium iodide for quantitative analysis (Vyas et al., 2013).Each ml of 0.1 N sodium thiosulphate ≡ 0.005585 g of ferric ion.Percentage magnetite content entrapped was found to be 53.In vitro drug release study of magnetic microspheres In vitro drug release study was performed by using dialysis bag diffusion method using phosphate buffer (pH 7.4) as dissolution media ( Saravanana et al., 2004).Table 4 and Figure 7 show in vitro release of magnetic microspheres.

RESULTS AND DISCUSSION
1. Percentage practical yield of the formulation was found to be 80.64% 2. Microspheres were found to have excellent flow properties.
3. Microspheres were found to be spherical in shape with average particle size of 20 µm.
4. Swelling ratio of magnetic microspheres was found to be increased with time. 5. Magnetic responsiveness of the microspheres was found to be more in the presence of magnetic field.6. Magnetic microspheres were found to be magnetically responsive.Percentage magnetite content entrapped in 6-TG loaded magnetic microspheres was found to be 53.7. Percentage drug release of magnetic microspheres was found to increase with time.

Conclusion
Particle size of magnetic microspheres was found to be 20 µm.It is considered that particles with size range of 10 to 100 nm are considered to be optimum for the drug delivery because they can easily escape the reticuloendothelial system.Microspheres are more stable as compared to other drug delivery systems such as nanoparticles, liposomes etc. However if they are made magnetic than they are, it is more advantageous as reticuloendothelial clearance can be minimized and target site specificity can be increased.Therefore the magnetic microspheres we have synthesized are promising candidates for successful drug loading and delivery to patients suffering from cancer.The drug release rates are also suitable for the drug delivery application.The main advantage of this technique is the reduction in the dose and effects of the drug.It is a challenging area for future research.

Figure 3 .
Figure 3. Graphical representation of swelling ratio of magnetic microspheres.

Figure 4 .
Figure 4. Apparatus for measuring magnetic responsivity of magnetic microspheres.

Figure 6 .
Figure 6.Graphical representation of microsphere content (%) retained in absence of magnetic field.

Table 2 .
Magnetic responsiveness of 6-thioguanine loaded microspheres in the presence of magnetic field

Table 3 .
Magnetic responsiveness of 6-thioguanine microspheres in absence of magnetic field.

Table 4 .
In vitro release of magnetic microspheres.