Temozolomide-Loaded PLGA Microspheres for Management of Glioblastoma Multiforme

Hosseinzadeh, Reihaneh (Department of Mechanical Engineering, University of Victoria)
Mirani, Bahram (Department of Mechanical Engineering, University of Victoria)
Pagan, Erik (Department of Mechanical Engineering, University of Victoria)
Pipaon Fernandez, Nahiane (Department of Mechanical Engineering, University of Victoria)
Akbari, Mohsen (Department of Mechanical Engineering, University of Victoria)


Glioblastoma multiforme (GBM) constitutes over half of 21,800 patients diagnosed with primary brain tumours in the United States. Complete surgical resection is almost impossible due to the infiltrating nature of GBM. Moreover, surgical resection is usually associated with damage to nearby healthy tissue. Therefore, the standard therapy for patients who are suffering from GBM is maximal safe surgery followed by radiotherapy plus concomitant and adjuvant chemotherapy with temozolomide (TMZ). Despite this aggressive treatment, the mean survival time for GBM patients is still low, about 14.6 months.

Conventional approaches for the treatment of GBM have numerous deficiencies. Firstly, the number of drugs that can be used for treating GBM are limited due to the challenges associated with crossing the blood-brain-barrier. Secondly, the systemic administration of chemotherapeutic agents, such as TMZ, which has a short half-life in plasma, should be done in high oral doses to achieve therapeutic levels. Furthermore, the prolonged oral administration of this chemotherapy drug has several side effects including nausea, vomiting, fatigue, headache, and lymphopenia. The shortcomings of systemic administration of chemotherapy together with the recurrence of gliomas in 90% of cases necessitate the development of a local drug delivery system.

Localized delivery of drugs directly to the tumour site has shown to be a promising method for overcoming the challenges associated with systemic delivery of drugs. Several studies have encapsulated TMZ in polymeric carriers using conventional oil-in-water (O/W) and water-in-oil-in-water (W/O/W) emulsion with the goal of sustained delivery of the drug over extended periods. However, the reported encapsulation efficiencies were extremely low (<5%) due to the amphiphilic structure of TMZ, which hampers the ability to scale up the process. Herein, we report a novel approach by which encapsulation efficiencies as high as 61% (a 12-fold improvement) have been achieved. We characterized the fabricated microparticles regarding their size and release profile of the drug as a function of particle size and polymer concentration. These particles hold great promise as injectable materials for the management of GBM.

Materials and Methods

Poly (D, L-lactide-co-glycolide) PLGA microspheres loaded with TMZ were fabricated with O/O emulsion procedure (Figure 1). The first oil phase (O1) was prepared by dissolving a known amount of PLGA and TMZ into acetonitrile. O1 was then emulsified with viscous liquid paraffin containing Span 80® used as the second oil phase (O2) by vortexing. Subsequently, the formed emulsion was continuously stirred at 55ºC for 2 hours to allow evaporation of the organic solvent. TMZ-loaded PLGA microspheres were also prepared by O/W and W/O/W emulsion. In order to determine the encapsulation efficiency, TMZ-loaded PLGA microparticles prepared with O/O emulsion were dissolved completely in acetonitrile. Dichloromethane was used for dissolving TMZ-loaded PLGA microspheres fabricated by O/W and W/O/W emulsion. The supernatant was then collected after multiple centrifugations and analyzed using a microplate reader. TMZ-loaded PLGA microspheres fabricated with different PLGA concentration were characterized by scanning electron microscopy (SEM). Commercially available ImageJ software was used to analyze the SEM images, thus determining the size of microspheres. In vitro release studies of polymeric microspheres were conducted in tris buffer in triplicate at 37 ºC. At specific time intervals, the withdrawn supernatant was analyzed using a microplate reader and replaced with fresh media.


The encapsulation efficiency of TMZ-loaded PLGA microspheres was optimized using different procedures and parameters (Table 1). We observed poor encapsulation efficiency for the microspheres prepared with O/W, O/W with TMZ saturation, and W/O/W methods because of the rapid diffusion of TMZ from the organic solvent to the water phase during the evaporation of the organic solvent and washing steps with deionized water. However, the microspheres fabricated with the same amount of TMZ and PLGA concentration by using the O/O emulsion demonstrated a high encapsulation efficiency of 47.70 ± 7.50 % due to the low solubility of TMZ in liquid paraffin. The analysis of SEM images revealed that an increase in PLGA concentration from 1.25 % to 10 % led to a rise in microsphere’s size (Figure 2A, B). A more viscous polymer solution after increasing the PLGA concentration accounts for obtaining larger microspheres. In vitro release studies indicated that larger polymeric microspheres have lower initial burst release and slower overall release rate due to their smaller surface-area-to-volume-ratio (Figure 2C).

Discussion and Conclusion

TMZ-loaded PLGA microspheres with high encapsulation efficiency were fabricated by using O/O emulsion solvent evaporation technique. The release kinetics of TMZ, as a diffusion-based phenomenon was tuned by the variation of the surface-area-to-volume ratio of fabricated microspheres. The proposed PLGA microspheres loaded with TMZ provides several advantages compared to conventional treatments of GBM including keeping the TMZ intact within the carrier, releasing the chemotherapy drug in a prolonged manner, minimizing the side effects associated with oral administration of TMZ, and enhancing the treatment efficacy by delivering the agent at the site of tumour.

Figure 1. Schematic of preparation of TMZ-loaded PLGA microspheres with O/O emulsion.

Table 1. Influence of different methods and parameters on the encapsulation efficiency of TMZ-loaded PLGA microspheres.

Figure 2. (A, B) SEM images and average size of TMZ-loaded PLGA microspheres prepared with different PLGA concentrations. (C) Release profile of microspheres fabricated with different PLGA concentrations.


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