Category: Formulation and Quality
Purpose: We have previously developed a strategy to render liposomes pH-sensitive: a protonation-induced conformational flip of lipid tails, using a trans-2-aminocyclohexanol moiety (TACH) as a molecular lever. Such pH-sensitive liposomes are called fliposomes. DOX-loaded fliposomes exhibited enhanced anticancer activity against 3D multicellular spheroids in vitro, which are more representative of solid tumors than 2D monolayer cells.
In this study, we aim to 1) establish an orthotopic animal model of breast cancer and 2) to evaluate the anticancer activity of fliposomes in the animal model.
Methods: Fliposomes containing lipids of various headgroups bearing a pH-sensitive conformational switch were prepared by film-hydration, freeze-thawing, and extrusion through polycarbonate membrane. The hydrodynamic diameter, ζ-potential, encapsulation efficiency and drug release at different pH were characterized.
For pharmacokinetics study, animals were anesthetized under isoflurane and blood sample was taken by cardio puncture. All blood samples were mixed with sterilized water at volume ratio 1:2 to lyse all blood cells. The samples were then stored at -20℃ until further analysis.
To establish the orthotopic breast cancer model, the MDA-MB-468 cells (8,000,000 cells/ mouse) were then injected into the mammalian fat pad on the second nipple around the left axillary region. Following inoculation, the mice were observed daily for general health and appearance of tumors.
After inoculation, the tumors grew for 2 weeks to 70 - 100 mm3. The tumor-bearing mice were randomly divided into five groups (control, doxorubicin solution, conventional liposome, MOR-C16 fliposome, and AZE-C16 fliposome) of eight mice each except for the non-treatment group (5 mice). The grouping was based on the tumor size.
Afterward, 5 groups of treatment were administered to the mice once a week for 4 weeks, while PBS (100 μL) was administered as a control group. The tumor volume was calculated using the formula: Tumor volume (mm3) = 0.5*L*S2
Where L the long axis and S is the short axis of the tumor lump. The tumors and hearts were removed and weighted when the animals were euthanized 63 days post the first dose.
Results: DOX-loaded AZE-C16 fliposome displayed non-linear pharmacokinetics following IV administration. Compared to the data listed in the literature, the pharmacokinetic parameters of AZE-C16 and the MOR-C16 fliposome were similar to free doxorubicin, suggesting that doxorubicin may have leaked prematurely from two fliposome during the circulation before accumulation at the tumor site.
Turkey's post-test comparisons between treatment groups showed that tumor volume of the MOR-C16 treatment group was significantly (p < 0.05) smaller than the free DOX treatment group from day 57 to 61, and significantly (p < 0.05) smaller than conventional liposome group at day 61. The tumor progression of fliposome (MOR-C16 and AZE-C16) treatment groups was delayed compared to free DOX and conventional treatment group, which suggested the pH-sensitive liposome improve the anticancer activity against TNBC on an orthotopic xenograft model. All the animals possessed steady body weight. The tumor weight was correlated with the tumor volume at the end of the experiment, confirming that the tumor volume measurement accurately reflected the tumor progression (Figure 2).
Conclusion: TNBC orthotopic xenograft model was established by injecting MDA-MB-231 cells into the murine mammalian fat pad, which simulates the tumor microenvironment. AZE-C16 and MOR-C16 fliposomes showed similar pharmacokinetic parameters. DOX-loaded fliposomes, especially the MOR-C16 fliposome, exhibited a significantly and a higher antitumor efficacy and delayed the cancer progression compared to free DOX and non pH-sensitive stealth liposomes.
Yifan Lu– University of the Pacific, Stockton, California
Zhongyue Yuan– stockton, California
Shen Zhao– Stockton, California
Zizhao Xu– Stockton, California
Yingbo Huang– University of the Pacific, Stockton, California
Mallika Vadlamudi– Stockton, California
Xinyu Pei– Stockton, California
Ruiqi Huang– Student, University of the Pacific, Stockton, California
Kevin Zhang– Stockton, California
Melanie Felmlee– Stockton, California
Xin Guo– University of the Pacific, Stockton, California