(T1430-03-17) Preparation and Evaluation of Temozolomide Solid Lipid Nanoparticles: Focus on Improvement in Bioavailability and Physiochemical Analysis for Decreased Toxic Effect in Glioblastoma Multiforme

Purpose: Glioblastoma (GBM) is the most severe type of brain tumor, with swift proliferation and infiltration of the surrounding brain tissue. Glioblastoma is currently treated with surgery, followed by radiotherapy and concomitant chemotherapy, usually with temozolomide (TMZ). Although quite extensive research has been conducted in recent years to establish a more efficient therapy approach for the management of GBM, the overall mortality of patients has not improved significantly. Various nano formulation approaches using liposomes, polymeric nanoparticles and nano structured lipid carriers have been explored. However, there exists several challenges such as poor encapsulation efficiency and non-scalability of the process. To overcome these challenges, it is suggested that a new formulation delivery that can address these challenges, be studied. It is suggested that solid lipid nanoparticles (SLNs) be used for the formulation of TMZ as SLNs outperform other polymeric nanoparticles in terms of drug loading capacity, drug retention, and particulate stability, resulting in improved overall bioavailability of loaded therapeutics to the target cell. Therefore, the present study employs the use of TMZ in SLNs vehicle through the nasal route for the effective treatment of GBM using nontoxic excipients such as glyceryl monostearate (GMS), glyceryl tri stearate (GTS), soya lecithin and Tween 80 (T80).

Methods: The solubility of TMZ was carried out in various lipid carriers. The TMZ loaded SLNs were prepared using high speed homogenization technique. Briefly, both aqueous and lipid phase were prepared separately. About 10 mg of drug was dissolved in the organic solvent that contained GMS and GTS. In a beaker containing 30 mL distilled water, T80 was added. To the organic phase, the aqueous phase was added under high homogenization, to solidify the lipid. The prepared TMZ-SLNs were stored in a refrigerator at 2±0.3°C for 6 hours. Characterization techniques were carried out using differential scanning calorimetry (DSC), powder X ray diffraction studies (PXRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), Zeta sizer for size and charge, cell line studies were carried out on GBM cell lines. In vitro release studies were carried out using the franz diffusion cell apparatus maintained at 37±0.5°C using simulated nasal fluid of pH of 6.5 and stirred at a speed of 50 rpm. The drug solution was placed into a cellophane membrane having 12,000 to 14,000 molecular weights cut off. The analytical samples were injected into an HPLC using methanol and acetic acid (0.25%) as mobile phase and quantified at 329.6 nm. Similarly, ex vivo studies on porcine nasal mucosa was performed after obtaining it from a local slaughter house. The tissue was washed using normal saline solution and was fixed on a franz diffusion cell with the serosal surface facing the donor side. The receptor compartment contained simulated nasal fluid and drug solution that was removed at predefined time points followed by injection into the HPLC system. Kinetic modeling was also performed on various mathematical models to understand the nature of drug release from the SLNs. Pharmacokinetic studies were performed using Sprague Dawley rats taking the approval from the institutional animal ethical committee (RCPT/IAEC/2021/08).

Results: TMZ loaded SLNs were successfully prepared by high-speed homogenization technique. The drug showed crystalline nature whereas the formulation was found to be amorphous with the absence of any sharp crystalline peak in the DSC thermogram. The result was supported by PXRD studies. It was further in concordance with the FESEM studies indicating better solubility. The size of TMZ-SLNs were found to be in the range of 121-167 nm respectively. The zeta potential was found to be +17.2±0.23 mV. The positive charge of the zeta potential was desired due to possible interaction with the negative charged mucosal layer. The entrapment efficiency was found to be 88±0.48%. Cell line studies indicated that the naïve drug had a higher IC₅₀ value which could imply that the formulation protected from systemic toxicity. In vivo and ex vivo studies indicated that the TMZ- SLNs had prolonged release as compared to naïve drug. Further, the S/L ratio and the amount of drug released after 4 and 24 hours were significantly correlated when drug release results were assessed using the Higuchi model. The mean residence time increased by 4.2 folds indicating better bioavailability of the drug.

Conclusion: TMZ has been reported for treating GBM. However, the niche formulation approach of increasing its bioavailability and entrapment efficiency has not been studied extensively. The present formulation could serve as a strategy to improve TMZ bioavailability. In a nutshell, the developed formulation using glyceryl monostearate (GMS), glyceryl tri stearate (GTS), soya lecithin and Tween 80 (T80) showed better mucus adhesion and bioavailability properties. The prepared formulation, could offer an aid in improving toxicity related aspects of TMZ.

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Acknowledgements: Authors are thankful to Central Instrumentation facility of Lovely Professional University for providing research support.

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