Category: Formulation and Quality
Purpose: The pH gradients in the tumor tissues represent a promising stimulus for triggered release. The extracellular pH (pHe) of normal tissue is around 7.4 while pHe in the tumor interstitium is 6.2 to 7.0. The pHe of the tumor core may be as low as 6.0.
Our group has developed imidazole-based convertible liposomes (ILC). The lipids with imidazole headgroups was protonated at acidic pH to turn convertible liposomes from stealth liposomes to cationic liposomes, therefore to enhance interaction with negatively charged tumor cells and release more drug.
This study is to prepare pH-sensitive imidazole-based convertible liposomes (ICL) and testify they suppress tumor cells more effectively compared with convertible liposomes.
Methods: The ICL contains 25% imidazole-based (DHI, DHMI, DHDMI) lipids, 70% DSPC and 5% DPPE-PEG. The ICL were prepared by the film-hydration, freeze-thawing and extrusion methods. The organic solvent of the lipid solution was evaporated at 60 ℃ to form a lipid film. Each lipid film was then hydrated in 300mM manganese sulfate solution prepared in buffer (pH 7.4, 5mM HEPES, 140 mM NaCl). The lipid suspension was then frozen by submergence into liquid nitrogen, followed by thawing in iced-water bath and 60℃ water bath. The resultant liposome suspension was extruded through polycarbonate membranes with 200 nm and 100 nm diameter pores. The liposome suspension was separated from the unencapsulated manganese sulfate and mixed with DOX (1mg/ml) to incubate for 1 h at 60⁰C. The mixed suspension was shaken with resin beads to remove the unencapsulated DOX from DOX-loaded liposomes. Sizes, encapsulation efficiency (EE), Zeta Potential and pH-triggered drug release were measured.
Hela and A549 cells were seeded in 96-well plates and incubated to reach diameter of 500nm to fabricate MCS tumor models. The Hela and A549 MCS models were treated with convertible liposomes (ICL-DHI, ICL-DHMI and ICL-DHDMI), compared with conventional liposomes and free DOX as the positive control. The anticancer efficacy can be demonstrated by the cell viability. Confocal images with fluorescence signal by DOX were captured to indicate the penetration of ICL.
Results: Liposomal formulations ICL were successfully prepared and characterized for physicochemical properties (Fig 1.). ICL showed increase in zeta potential from pH 7.4 to pH 6.0 while conventional liposomes didn’t show significant change, which indicated the protonation of ICL, to turn from stealth liposomes into cationic liposomes at pH 6.0 (Fig 1.). The drug release results indicated ICL, especially ICL-DHMI, released more drug at pH 6.0 compared with pH 7.4. (Fig 1.).
The cytotoxicity result indicated ICL, especially ICL-DHMI, suppressed the cell viability of Hela MCS more effectively compared with convertible liposomes (Fig 2.).
Conclusion: Triggered by acidic pH, ICL release more payload drug, as well as inhibit the growth of Hela MCS more effectively, compared with convertible liposomes. In the future, morphology and MCS penetration result of ICL will be obtained. The cytotoxicity on A549 MCS by ICL will be certified.
Vijay Gyanani– Stockton, California
Xinyu Pei– Stockton, California
Zhongyue Yuan– stockton, California
Zizhao Xu– Stockton, California
Yifan Lu– University of the Pacific, Stockton, California
Yingbo Huang– University of the Pacific, Stockton, California
Xin Guo– University of the Pacific, Stockton, California