Abstract A118: Development and evaluation of novel herceptin immunoliposome encapsulating cisplatin against HER2-overexpressing cancer cell.
Authors: Okada Masashi , Nakano Takafumi , Hirai Masahiko , Chen Ling , Li Yue-Guang , Mizutani Akifumi , Kudoh Takayuki , Igarashi Koichi , Seno Masaharu 
Published: June 2012

Introduction: Liposomes, a number of designs have been applied to confer them specific affinity to tumor sites, are considered as appropriate carriers to delivery anti-cancer agents to tumor sites so as to reduce side effects. Breast cancer is one of disease with large numbers of patient all over the world. HER2/neu overexpressed breast cancer is malignant and poor prognostic. Targeting therapy of HER2 is required to overcome such a critical cancer. Herceptin (Trastuzumab) is antibody drug for HER2 overexpressed cancer. We exploited Herceptin to enhance targeting ability of liposome and anti-cancer activity by using cis-diamminedichloro-platinum (II) (CDDP) encapsulating liposome. We have previously developed efficient procedure of encapsulation of CDDP. Although it has long been a substantial problem to prepare liposomes loading high quantity of CDDP due to its insolubility in aqueous phase, our procedure enabled the loading efficiency hundreds more than those reported previously. In this study, these techniques were combined together to prepare liposomes conjugated with Herceptin loading CDDP to target breast cancer in vivo without unwanted side effects due to the toxicity of CDDP.

Methods: Liposomes were prepared by improved cholate dialysis method. The lipids mixed in methanol/chloroform solvent were evaporated, followed by dissolved in solution of cis-diamminedinitratoplatinum (II) (CDDP3), one of the various CDDP derivatives with of high water solubility and is readily converted into CDDP in the presence of chloride ions, and then dialyzed against sodium chloride. Herceptin was conjugated with CDDP pre-loaded liposomes by cross-link agent DTSSP.CDDP encapsulated liposome and Herceptin conjugated immunoliposome were evaluated for anti-cancer activity in vitro by MTT assay. Targeting ability of immunoliposome was confirmed by confocal laser scanning and by in vivo imaging of BT474 xenografted model to confirm whether liposomes may accumulate at the tumor region. BT474 human ductal carcinoma was xenografted to estradiol capsule pre-implanted Balb/c (nu/nu) female mice. Drugs were administered to mice at dose of 20 mg/kg of mice body weight. The tumor sizes were monitored and survival rate of drug administered mice are calculated to evaluate whether drug administration contributed to prolong life of mice.

Results: The peak sizes of liposomes were approximately 200 nm, whose sizes are suitable to accumulate in the tumor region via enhanced permeation and retention (EPR) effect. Though CDDP sensitivity of BT474 was less than other cell lines such as SKBr3, SKOv3 and MDA-MB-231, encapsulation of CDDP into liposome enhanced anti-cancer activity for BT474. Though fluorescence from liposomes encapsulated with Cy5.5 was detected from tumor of mice administered both normal liposome and Herceptin-liposome, mean tumor volume of the mice treated with Her-CDDP-Lip got smaller than that of the mice treated with CDDP-Lip. All mice treated with CDDP alone died within 4 days, while fifty percent of mice treated with Her-CDDP-Lip and CDDP-Lip survived up to the end of experiment.

Conclusions: The Herceptin liposome was with strong anti-cancer activity both in vitro and in vivo when compared to usage of CDDP alone. We developed new combination therapy of Herceptin with CDDP, not common for treatment of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A118.