Multifuntional Liposomal Drug Delivery System For Treating Bone Metastasis From Prostate Cancer

Objective In order to investigate how to improve therapeutic efficacy in a mice model of bone metastasis from prostate cancer, a liposomal drug delivery system conjugated with cyclic arginine-glycine-aspartic acid peptide(cRGDyk) and a thermosensitive liposome were thus developed.Methods(1) The morphology and size of the RGD-CIS-liposome were observed on a transmission election microscope(TEM). The hydrodynamic diameter, polydispersity(PDI), and zeta potential of the liposomes were determined by dynamic light scattering(DLS). The drug encapsulation percentage(EP %), loading efficiency(LE %) and the cumulative release of cisplatin from the liposomes were determined by a HPLC method.Targeting efficiency of the liposomes was investigated by flow cytometry and confocal laser scanning microscope. Cell viability was analyzed by the use of MTT after RM-1 cells were treated with various concentration of drugs. C57BL/6 mice were intravenously administered via the tail vein with free cisplatin,CIS-liposomes, and RGD-CIS-liposomes, plasma and tissue samples were analyzed through HPLC system. Fluorescence intensity in tumor site was measured by IVIS imaging softwarefollowing RM-1 cells injection from tibia of right hind limb. The pain-related behaviors of the mice were tested at 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, and 28 days following tumor cells injection.Body weight of tumor-bearing mice in each group was recorded throughout the experiments and the mice were checked for survival analysis every day. Whole-body CT assessment was performed with tumor-bearing mice on a CT scanner at 22 day following injection of RM-1 cells. At 2, 7, and 14 day after injection of PBS,cisplatin, CIS-lipsomes, and RGD-CIS-liposomes, the liver and kidney were harvested and stained with haematoxylin and eosin(H&E) for histology study.(2) The morphology and size of the thermosensitive liposomes(TSL) were observed on a TEM. The hydrodynamic diameter, polydispersity(PDI), and zeta potential of the liposomes were determined by DLS. The drug EP % and the cumulative release of DOX Doxorubicin from the liposomes were determined by fluorescent quenching property. Cell viability were analyzed by the use of MTT after RM-1 cells were treated with various concentration of drugs with or without NIR laser irradiation.Cellular uptake of TSL was determined by using the fluorescence of DOX itself. Living cell imaging was investigated by Calcein AM. The pain-related behaviors of the mice were tested at 1, 3, 7,14, and 21 days following tumor cells injection. Body weight of tumor-bearing mice in each group was recorded throughout the experiments and the mice were checked for survival analysis every day. Whole-body CT and X-ray assessment were performed with tumor-bearing mice on a CT scanner at 22 day following injection of RM-1 cells. At 7, and 14 day after injection of PBS, DOX, Au,DOX-Au and DOX-TSL-Au, the heart, liver and kidney were harvested and stained with H&E for histology study.Results(1) RGD-CIS-liposomes showed uniform sphericalmorphology and exhibit representative vesicular structure. The liposomes perform low EP % but high LE % with decreased weight ratio of liposome to cisplatin(increased cisplatin concentration). The results of CLSM images and flow cytometry suggested that more cRGDyk conjugated liposomes are more easily internalized by the cells. RGD-CIS-liposomes resulted in further higher cytotoxicity by MTT assays. Cisplatin had a short biological half-life and was rapidly cleared from the systemic circulation. The high accumulation of three drugs was found in the liver and spleen. Cisplatin group was found to be significantly decreased in drug level over time, while CIS-liposomes and RGD-CIS-liposomes groups remained high drug level in all organs at 24 h post-injection, resulting from long retention time of the liposomes. IVIS results indicated the accumulation of two formulations in the tumor sites in the bone, but the accumulation of cRGDyk conjugated liposomes in the tumor was higher than that of the liposomes without cRGDyk conjugation at all time points. It was found that although cisplatin, CIS-liposomes, and cRGDyk treatments can relieve pain compared with control group, there was no significant difference among them at any time. Comparatively,spontaneous lifting behavior of RGD-CIS-liposomes group occurs at 16 day and lifting time remains at a low level. Moreover, a significant difference was observed between RGD-CIS-liposomes group and other four groups, especially at 18, 20, and 22 day. No changes in body weight were observed throughout the experiments.The results of CT showed that RGD-CIS-liposomes could inhibit bone destruction. Although cisplatin treatment showed a certain degree of histopathological abnormalities or lesions in the liver at14 day, no noticeable toxicity was observed in CIS-liposome and RGD-CIS-liposomes grops.(2) TSL had a rapid release of drug in application of mildhyperthermia and was stable at room temperature. TSL could increase drug release under mild hyperthermia in RM-1cells. HE suggest that DOX treatment showed a certain degree of histopathological abnormalities or lesions in the liver and heart at14 day.Conclusions(1) cRGDyk conjugated liposomes showed enhanced therapeutic efficacy in treating bone metastasis including tumor regression, pain relief, and overall survival improvement, by taking advantages of targeted drug delivery and synergistic antitumor activity of cRGDyk and loaded drug.(2) Mild hyperthermia(43?) plus chemptherapy can inhibit the proliferation of prostate cancer cells.