Purpose: A hypoxia-activated prodrug (HAC) has been developed for the treatment of chemotherapy-resistant tumor. Several obstacles of HAC have been found in formulation development, such as short half-life in bodies and unfavorable physical characteristics of low solubility and low partition coefficient. To overcome these obstacles, nanostructured lipid carrier (NLC) was utilized to incorporate HAC. NLC is one of the lipid-based drug delivery systems consisting of solid lipids, liquid lipids, and surfactants with particle sizes in the nanometer range. NLC possesses numerous advantages, such as high loading capacity and avoidance of hazardous organic solvents during the manufacturing process. Most importantly, NLC could improve solubility, pharmacokinetics and tumor accumulation for the drugs encapsulated. In this study, the NLC strategy was adopted through the cold emulsification method to enhance the entrapment efficiency of HAC. To find out the composition of NLC, lipids, and surfactants were selected and tested for the formulation, followed by evaluating the particle size, morphology, polydispersity index (PDI), drug degradation and long-term storage stability. In addition, in vitro chemosensitivity study was performed to examine the cytotoxicity under hypoxia condition (1% O2).
Methods: The mixture of lipid phase containing palmitic acid, castor oil, lecithin and HAC was heated up to 80℃ and mixed homogeneously. For cold emulsification method, the hot lipid phase was poured into liquid nitrogen for solidification. The cold solid lipid phase was then ground into lipid powder and added to the aqueous phase containing surfactants and water. The solution was emulsified through probe sonication in ice water bath until the microemulsion was formed. On the other hand, for hot emulsification method, the 80℃ lipid phase was emulsified with the 80℃ water phase. Afterward, the microemulsion was diluted with 4℃ PBS at a volume ratio of 1:5 following homogenization for 20 minutes. NLC-HAC drug degradation study was conducted with HPLC. The entrapment efficiency, particle size and PDI of NLC were evaluated for 30 days. For chemosensitivity study, cells were loaded into 96-well plates and incubated under normoxia and hypoxia conditions overnight. On the next day, the medium was replaced with medium containing free-form or NLC-HAC at a range of concentrations. After 96-hour incubation, the medium was removed and replaced with 0.4 mg/mL MTT reagent and incubated for 2 hours.
Results: The solubility of HAC in castor oil (2 mg/mL) was significantly higher than oleic acid (0.8 mg/mL); therefore, castor oil was chosen to be the liquid lipid of NLC. Tween80 and Kolliphor ELP were tested for those lipids based on their HLB and they showed lower cytotoxicity compared to Tween20. HAC loaded NLCs were successfully prepared using both the hot and cold emulsification methods. The entrapment efficiency and loading capacity of NLCs for HAC manufactured through cold emulsification method were 26.63 ± 1.59% and 0.404 ± 0.039 %, respectively (Table 1). Meanwhile, through hot emulsification method, the entrapment efficiency and loading capacity were 14.36 ± 3.44% and 0.220 ± 0.045%, respectively. The lower efficiency and capacity in hot emulsification method may be due to the high temperature which increased the molecular kinetic energy of HAC from lipid phase that led to rapid partitioning to the aqueous phase. Stability test indicated that in 7 days, there was no significant degradation of HAC in NLCs prepared by the cold emulsification method. Storage stability under room temperature showed no difference of entrapment efficiency, particle size and PDI for 30 days (Fig. 1). Chemosensitivity study demonstrated similar extent in cytotoxicity using either free-form HAC or NLC-HAC under hypoxia condition (1% O2) at the concentration of 1.5 μM (Fig. 2).
Conclusion: Our data supported that NLC-HAC prepared by cold emulsification method was stable with good entrapment efficiency and loading capacity, and chemosensitivity study showed similar cytotoxicity efficacy to free-form HAC, indicating the possibility of using NLC to overcome the current obstacles for HAC.
Po-Chun Peng– postdoctoral researcher, Department of Biochemical Science and Technology, National Taiwan University
Chin-Tin Chen– Professor, Department of Biochemical Science and Technology, National Taiwan University