Temozolomide/PLGA microparticles and antitumor activity against Glioma C6 cancer cells in vitro

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Abstract

The purpose of the present study was to develop implantable poly(d,l-lactide-co-glycolide) (PLGA) microparticles for continuous delivery of intact 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide (temozolomide, TM) for about a 1-month period and to evaluate its cytotoxicity against Glioma C6 cancer cells. The emulsifying-solvent evaporation process has been used to form TM-loaded PLGA microparticles. The influences of several preparation parameters, such as initial drug loading, polymer concentration, and stirring rate were investigated. Scanning electron microscopy (SEM) showed that such microparticles had a smooth surface and a spherical geometry, i.e. microspheres. The differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD) results indicated that TM trapped in the microparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The release profiles of TM from microparticles resulted in biphasic patterns. After an initial burst, a continuous drug release was observed for up to 1 month. Finally, a cytotoxicity test was performed using Glioma C6 cancer cells to investigate the cytotoxicity of TM delivered from PLGA microparticles. It has been found that the cytotoxicity of TM to Glioma C6 cancer cells is enhanced when TM is delivered from PLGA polymeric carrier and, PLGA only did not affect the growth of the cells. Meanwhile, the cytotoxic activity of TM powder disappeared within 12 h.

Introduction

A major factor limiting intracranial therapeutic levels of systemically administered chemotherapeutic agents is the physiologic barriers of the brain (Gallia et al., 2005). The presence of the blood–brain barrier (BBB) restricts permeability of certain drug molecules within the brain and prevents diffusion of these agents into the brain tumor (Kornblith and Walker, 1988). Temozolomide(3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide, TM) is one of the most effective antineoplastic agents for malignant glial tumor, partially due to its ability to cross the BBB (Reni and Mason, 2004). However, TM must be administered in high systemic doses to achieve therapeutic brain levels due to its short half-life of about 1.8 h in plasma (Baker et al., 1999). Furthermore, prolonged systemic administration is associated with some side effects such as nausea, vomiting, fatigue and headache.

Recent reports have shown that polymeric devices implanted into the brain can release locally neuroactive substances for extended periods of time (Menei et al., 1996, Menei et al., 1997). In this manner, the brain implantation of polymeric devices has been achieved in men for the treatment of malignant cerebral tumors (Brem et al., 1995). Poly(d,l-lactide-co-glycolide) (PLGA) is a well-known biodegradable polymer, which has long history of safe use in pharmaceutical and medical applications (Hutchison and Furr, 1990). Furthermore, the study have demonstrated the biocompatibility and biodegradability of blank poly(d,l-lactide-co-glycolide) (PLGA) microspheres implanted in the brain tissue (Emerich et al., 1999, Menei et al., 1993).

The present research report describes the in vitro release of TM from PLGA-based microparticles fabricated using the emulsifying-solvent evaporation method. The influences of several preparation parameters, such as initial drug loading, polymer concentration, and stirring rate were investigated. The physical characteristics of TM-loaded PLGA microparticles were studied using scanning electron microscopy (SEM), powder X-ray diffraction (XRD), and differential scanning calorimetry (DSC). The in vitro antitumor activity of TM released from the microparticles was assessed against Glioma C6 cancer cells in comparison with that of TM powder.

Section snippets

Materials

PLGA (75:25 mole ratio of lactide to glycolide) having molecular weight of 20,000 g/mol were purchased from Shandong Medical Equipment Research Institute (China). TM was supplied by Friend Pharmaceutical Co. Ltd. (Beijing, China). Polyvinyl alcohol (PVA) (88% hydrolyzed, Beijing, China) was used as the emulsifying agent. The methylene chloride (CH2Cl2) was used without further purification (Shanghai, China). All other chemicals were reagent grade. Deionized water was prepared by a Milli-Q

Microparticle preparation

TM-loaded PLGA microparticles were prepared using emulsifying-solvent evaporation method. In the process, aqueous phase containing 2% (w/v) PVA was saturated with TM beforehand to improve the encapsulation efficiency of TM. It was found that when aqueous phase was not saturated with TM, TM in the organic phase was almost diffused into the aqueous phase and the encapsulation efficiency was only about 30%. The encapsulation efficiency was significantly improved when aqueous phase was saturated

Conclusions

TM-loaded PLGA microparticles were prepared by emulsifying-solvent evaporation method in reproducible manner. Several preparation parameters, such as initial drug loading, polymer concentration, and stirring rate played a predominant role in the preparation. Microparticles had spherical shape, i.e. microspheres. From SEM, X-ray and DSC results, it appeared that TM trapped in the microparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The release profiles

Acknowledgements

The authors are very grateful to Professor Zhi-Jian Yue for many helpful discussions and technical support. They also express their appreciation to Professor Ding-Jian for providing the C6 glioma cell line samples.

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