Multifunctional SPIO/Mitoxantrone-loaded Liposomes For Cancer Therapy And MR Imaging

Cancer is a life threatening disease. Liposomes are emerging as a highly integrated nanoplatform for cancer targeting, drug delivery and tumor imaging applications. In this study, we described a multifunctional liposome system that is target to a luteinizing hormone-releasing hormone(LHRH) receptor, and encapsulated with superparamagnetic iron oxide (SPIO) and mitoxantrone (MTX) for magnetic resonance imaging(MRI) and therapeutic delivery, respectively. The research consists of four major sections:1. The preparation and characterization of LHRH receptor target liposomes loaded with SPIO/mitoxantrone SPIO loaded liposomes were prepared by lipid film hydration and an ultrasound dispersion process. MTX was loaded with transmembrane ammonium sulfate gradient. LHRH analogs, gonadorelin, with affinity for the LHRH receptor was chemically coupled to the distal end of polyethylene glycol of liposomes via post insertion technique. The liposomes were characterized in terms of its size, ligand density, drug loading, and leakage properties. The results indicated that the size of the liposomes was in the range of 120-150 nm. More than 70% of the gonadorelins were inserted into the liposome bilayer. Up to 90% loading efficiency of SPIO and MTX were achieved. The in vitro drug release experiments showed that the liposomal formulations exhibited sustained drug release characteristics.2. In vitro intracellular uptake and anti-tumor effect of LHRH receptor target multifunctional liposomes loaded with SPIO/mitoxantrone The targeting nature and anti-tumor effect of the liposomes were evaluated in vitro using cultured LHRH receptor high expressing MCF-7 breast cancer cells and LHRH receptor low expressing SK-OV-3 ovarian cancer cells. The MCF-7 cells showed higher uptake to LHRH receptor target SPIO/MTX loaded multifunctional liposomes (LHRH-MTX-MLs) than untarget SPIO/MTX loaded liposomes(MTX-MLs). While SK-OV-3 cells displayed no obvious different in the uptake of the two liposomal formulations, indicating that gonadorelin-modified liposomes had significantly higher affinity to the LHRH receptor. The in vitro antitumor showed that, after treatment with MTX-MLs or LHRH-MTX-MLs at concentration of 300 ng/ml MTX for 120 hours, the viability of MCF-7 cells were (45.3±2.3)% and (54.8±1.2)%, respectively. LHRH-MTX-MLs exhibited a better anti-tumor efficiency than MTX-MLs (P<0.05).3. Pharmacokinetics, biodistribution and anti-tumor effect of LHRH receptor target liposomes loaded with SPIO/mitoxantrone Plasma pharmacokinetic analyses of two liposomal MTX formulations and free MTX were performed in normal rats, the t1/2βof LHRH-MTX-MLs, MTX-MLs and MTX were 13.5 h,11.1 h and 0.33 h, respectively. Compared with free MTX, both LHRH-MTX-MLs and MTX-MLs could enhance the retention of MTX in plasma. Biodistribution studies of two liposomal MTX formulations and free MTX were performed in tumor bearing BALB/c mice, Compared with free MTX, both LHRH-MTX-MLs and MTX-MLs preferentially accumulated into tumor, liver, spleen etc. In contrast, the drug concentration in heart was significantly decreased. The antitumor efficacy of the liposomes was evaluated in BALB/c mice with breast cancer. LHRH-MTX-MLs showed significantly decreased tumor volumn in comparison with free-MTX and MTX-MLs in the mice models. At day 21, the inhibition rates of LHRH-MTX-MLs, MTX-MLs and MTX were (47.3±4.6)%, (42.5±5.9)% and (25.2±3.7)%, respectively, demonstrating that LHRH-MTX-MLs, the specific LHRH receptor targeted liposomes, could more efficiently inhibit tumor growth and deliver anticancer drug into tumor cells than free MTX and MTX-MLs(P<0.05).4. In vitro and in vivo MRI contrast effect of LHRH receptor target liposomes loaded with SPIO/mitoxantrone As a MRI contrast agent, the SPIO could make the signal weaken, by which to differentiate cancerous tissue from surrounding benign tissues. In vitro MRI experiments showed that the MRI signal intensity of LHRH-MTX-MLs and MTX-MLs decreased according to the iron concentration in the T2-weighted images, revealing that both LHRH-MTX-MLs and MTX-MLs displayed T2 enhancement in vitro. The MRI experiments on tumor bearing mice showed that both LHRH-MTX-MLs and MTX-MLs can decrease the tumor signal intensity value. Moreover, the tumor signal intensity value after an injection of LHRH-MTX-MLs was decreased significantly compared to MTX-MLs, according to the enhanced T2-weighted images(P<0.05).In conclusion, we have developed of LHRH receptor target multifunctional liposome loaded with SPIO/mitoxantrone, LHRH-MTX-MLs. The results demonstrated that LHRH-MTX-MLs had significantly high affinity to their target cells and better antitumor efficiency. Meanwhile, LHRH-MTX-MLs also displayed excellent T2 enhancement in vitro and in vivo, and could used as MRI contrast agents to differentiate cancerous tissue from surrounding benign tissues. In short, LHRH receptor target multifunctional liposomes loaded with SPIO/mitoxantrone is a promising candidate for treating cancer, tumor MR molecular imaging and monitoring the progress of the cancer using MRI.