Magnetite nanoparticle-loaded anti-HER2 immunoliposomes for combination of antibody therapy with hyperthermia
Introduction
Hyperthermia is a promising approach to cancer therapy [1], [2]. The inevitable technical problem with hyperthermia is the difficulty in heating only the local tumor region to the intended temperature without damaging the surrounding healthy tissue. Magnetite nanoparticles have been used for hyperthermia treatment in an attempt to overcome this obstacle [3], [4]. If magnetite nanoparticles can be made to accumulate only in tumor tissue, cancer-specific hyperthermia can be achieved by generating heat in an alternating magnetic field (AMF) due to hysteresis loss [5].
We have developed magnetite cationic liposomes (MCLs) as mediators of intracellular hyperthermia [6], [7], [8]. These cationic liposomes exhibit improved adsorption and incorporation into tumor cells, and have 10-fold higher affinity for tumor cells than neutrally charged magnetoliposomes [6]. We previously demonstrated the efficacy of MCL-mediated hyperthermia in animals with several types of tumors, including B16 mouse melanoma [9], T-9 rat glioma [8], Os515 hamster osteosarcoma (unpublished results), and VX-7 squamous cell carcinoma in rabbit tongue [10]. Although MCL-mediated hyperthermia was found to be very effective for inducing complete regression of tumors, these cationic MCLs must be directly injected into the tumor tissue. From this background, we developed antibody-conjugated liposomes (immunoliposomes) containing magnetite nanoparticles. We previously constructed immunoliposomes using mouse G22 monoclonal antibody (MAb) against human glioma cells [11] and mouse G250 MAb against human renal cell carcinomas [12], and demonstrated their tumor-specific targeting ability using animal models.
Human epidermal growth factor receptor-2 (HER2), which is overexpressed in 20–30% of breast cancers and is expressed at low levels in certain normal tissues [13], has been identified as a possible target for antibody-based therapy based on the following two considerations. First, because HER2 overexpression contributes to tumor progression, anti-HER2 antibody may interfere with this important mediator of tumor growth. A murine anti-HER2 MAb, muMAb4D5, inhibits growth of HER2-overexpressing breast cancer cells in vitro [14] and in vivo [15]. A humanized modifier of this antibody, Herceptin (Trastuzumab), which retains these properties while reducing the potential for immunogenicity [16], is currently in clinical use. Second, the stable overexpression of HER2 on the tumor cell surface provides an ideal target antigen for a drug delivery system (DDS). Anti-HER2 immunoliposomes have been developed as a tumor-targeting vehicle; they specifically bind to and become incorporated into HER2-overexpressing tumors. Anti-HER2 immunoliposomes have a dual role in breast cancer therapy: as a DDS for cytotoxic drugs; and as a vehicle for Herceptin, which has antitumor activity. Herceptin has antiproliferative effects when administered by itself, and is also efficacious when combined with chemotherapy [17]. Park et al. reported that anti-HER2 immunoliposomes containing doxorubicin targeted HER2-overexpressing tumor cells, combining the cytotoxic effects of Herceptin with those of doxorubicin [18].
In the present study, we constructed anti-HER2 immunoliposomes containing magnetite nanoparticles and investigated the feasibility of using them for combined anti-HER2 antibody therapy and tumor-specific hyperthermia. To the best of our knowledge, this is the first time that the combination of anti-HER2 antibody therapy and tumor-specific hyperthermia has been shown to exhibit a strong cytotoxic effect.
Section snippets
Cell culture and antibodies
SKBr3 human breast cancer cells with high HER2 expression (American Type Culture Collection) were cultured in McCoy's 5a medium (Gibco BRL, Gaithersburg, MD) supplemented with 1.5 mM l-glutamine, 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 0.1 mg/ml streptomycin). MDA-MB-231 human breast cancer cells with low HER2 expression (American Type Culture Collection) were cultured in Leivovitz's L-15 medium (Gibco BRL), supplemented with 2 mM l-glutamine, 10% FBS and
Combined effect of anti-HER2 antibody and hyperthermic treatment on SKBr3 cells
Fig. 1 shows relative cell number of SKBr3 cells on the 8th day after hyperthermic treatment at 42.5 °C for 60 min. For hyperthermic treatment alone, relative cell number was 42.5±6.3%. When hyperthermic treatment was combined with simultaneous Herceptin treatment (2.5 μg/ml), the relative cell number decreased to 19.6±4.1%. In contrast, when hyperthermic treatment was combined with Rituxan (control antibody), no significant decrease in relative cell number was observed. To assess the influence
Acknowledgements
The authors would like to thank Toda Kogyo Co. for supplying the magnetite. This work was partially supported by a Grant-in-Aid for Scientific Research (No. 13853005), University Start-Ups Creation Support System, and the 21st Century COE Program ‘Nature-Guided Materials Processing’ from the Ministry of Education, Science, Sports and Culture of Japan.
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