(W1030-03-18) Effect of Phospholipase A₂(PLA₂) Enzyme on Temozolomide Release from Composite Nanovesicles

Purpose: Vesicle systems such as niosome and liposome with bilayer membranes and interior cavities are favored in the drug delivery design due to high drug loading capacity, desirable drug release properties, wide surface area, and the ability to incorporate hydrophilic and lipophilic components. phospholipase A₂ (PLA₂) enzyme is a group of enzymes that can degrade the phospholipids membrane through hydrolysis reactions. The goal of this study is to evaluate the effect of PLA₂ enzyme on the release behavior of temozolomide (TMZ) from composite nanovesicles.

Methods: The niosomal and liposomal formulations were prepared through the thin film hydration method. In niosomal composition, predetermined amounts of nonionic surfactant (Span 60), cholesterol, soybean lecithin, and chloroform were mixed. Chloroform was then evaporated by rotary vacuum evaporator at 60-65 ºC to result in a thin film. The thin film was then hydrated with the mixture of TMZ, and phosphate buffered saline (PBS, pH=7.4) at 60-65 ºC to form the niosomal vesicles. To prepare the liposomal nanovesicles, predetermined amounts of dipalmitoylphosphatidylethanolamine (DPPE), soybean lecithin, and chloroform were used via the same method mentioned above. The drug entrapment efficiency is then determined based on the free drug content in supernatant upon centrifugal filtration. Briefly, the nanovesicles suspensions were centrifuged for 30 mins at 4 °C and at a speed of 4500 rpm. After washing steps, the supernatant absorbance was measured to obtain the free drug concentration using a UV−vis spectrophotometer at 260 nm. The percentage of drug entrapment in the niosomal and liposomal carriers was then calculated. In vitro drug release of TMZ-nanovesicles was conducted using a dialysis method in PBS as a release medium at pH around 7.4 under sink condition. Briefly, 1 mL of the niosomal and liposomal suspensions were poured into separate dialysis tubes (MWCO 8-10kDa) that contain different concentrations of PLA₂ (0 µg/mL (Control), 0.6 µg/mL and 3 µg/mL). The release study was performed at 37 °C with 50 mL of the medium and, a stirring speed of 60 rpm. Samples were withdrawn from the release medium at predetermined time intervals (30 mins,1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h) and replaced with fresh media. Samples were then analyzed for TMZ concentration using UV–Vis spectrophotometer at a wavelength of 260 nm. The cumulative drug release in PBS was calculated for up to 24 hours for both niosomal and liposomal nanovesicles. Then the release behavior of TMZ-loaded niosome/liposome in the presence and absence of PLA₂ enzyme was evaluated.

Results: The drug entrapment efficiency for niosome and liposome was around 88.11± 2.96% and 80.53±1.14%, respectively. As per the in vitro release study, TMZ-loaded niosome and TMZ-loaded liposome without PLA₂ (control) demonstrated sustained-release behavior and it was found by 85.53±11.5% and 98.99±4.92%, respectively for up to 24 hours (Figure 1). As shown in Figure 2, in the presence of 3 µg/mL and 0.6 µg/mL PLA2, 99.39±0.82% and 77.47±5.47% of TMZ-loaded liposome were released respectively within the first 6 hours, while these numbers decreased to 40.77±3.80% and 37.78±2.69% in the TMZ-loaded niosome. Slower drug release from niosome over the liposome was significantly observed. This may be attributed to the niosomal composition that may cause slower drug release due to more resistance to PLA₂ degradation enzyme confirming that PLA₂ enzyme was responsible for the rapid drug release in liposome. In the presence of different PLA₂ enzyme concentrations, TMZ-loaded niosome demonstrated sustained-release behavior with no significant differences compared to the control, while the TMZ-loaded liposome exhibited immediate-release behavior within the 6 hours with significant differences compared to the control (Figure 3).

Conclusion: This work was to investigate the role of PLA₂ enzyme in degradation of composite nanovesicles and release behavior of TMZ-loaded niosome/liposome. In this study, the presence of PLA₂ enzyme, either in high or low concentrations, accelerated the release of TMZ from the liposome by damaging the integrity of the liposome due to enzymatic degradation of phospholipids.

Acknowledgements: This research was supported in part by Nova Southeastern University Health Professions Division Grant # 334632.

Figure 1. Cumulative drug release from niosome and liposome in the absence and presence of different PLA2 enzyme concentrations for up to 24 hours. Niosome and liposome without PLA2 enzyme served as controls. Data represents ± SD (n = 3).

Figure 2. Comparison of the drug release between the niosomes and liposomes in the absence and presence of different PLA2 enzyme concentrations during the first 6 hours. Niosome and liposome without PLA2 enzyme served as controls. Data represents ± SD (n = 3). *P < 0.05, ns P>0.05.

Figure 3. Comparison of the drug release between the control and different PLA2 enzyme concentrations in each group during the first 6 hours. Data represents ± SD (n = 3). *P < 0.05, ns P>0.05.