Long Circulationg Liposomes Mediated Internal Radiation And Antisense Therapy For Breast Cancer

OBJECTIVE (1) To investigate the preparation of ¹25I/131I-oligonucleotide (ON) and 125I/131I-ON long circulating liposomes (LCL). (2) To describe the functional evaluation of phosphorothioate antisense oligonucleotides (ASON) with bispecificity for bcl-2 and bcl-xl compared with bcl-2 ASON. (3) To evaluate and compare the combined therapeutic effects of antisense and internal radiation in vitro and vivo mediated by anionic LCL (NA), cationic LCL (PA), neutral-LCL (A-D), and conventional liposomes (CA). METHODS (1) ON was labeled with radioiodine using thallium chloride tetrahydrate (TIC1₃) as an oxidizing agent. The reaction conditions were optimized after systematic variation of pH, reaction time and reaction temperature. ¹25I/¹31I-ON was separated from free radioiodine by Sephadex G25 column chromatography. The radiolabeling efficiency, radiochemistry purity, specific radioactivity and stability of 125I-ON in 0.01M HEPES buffer and human serum were observed respectively. (2) PC(egg-phosphatidylcholine), CH(cholesterol),DSPE-PEG2000 (distearyl phosphatidylethanolamine-polyethylene glycol, molecular weight 2000), DPPG (dipalmitoylphosphatidylglycerol) and DDAB (dimethyldioctadecyl-ammonium bromide) in the molar ratios of 0.50: 0.35: 0.06: 0.10, 10:4:1:3.3, 2:1:0.2:0.66 and 0.50: 0.29: 0.06: 0.12 were dissolved in different organic solvents such as iso-propyl ether, chloroform, methanol with varing ratios of volume to volume. Then 125I-ON-liposomes were prepared by the means of modified reverse-phase evaporation, and their quality was evaluated after the crude liposomes were repeatedly extruded through 400nm, 200nm, 100nm polycarbonate membranes consecutively. (3) The extent of cell uptake of liposome formulations and free 125I-ASON (FA) was evaluated using MCF-7 cell line.(4) The pharmacokinetics and tissue distribution were studied in rabbits and normal mice after the iv administration of different liposomal preparations and FA. Tissue distribution of NA and FA was investigated in virgin emale Sprague-Dawley (SD) rats with n-methyl nitrosourea (MNU) -induced in situ breast cancers. (5) HE staining, methythiazolyltetrazolium (MTT) and flow cytometry (FCM) were adopted to evaluate the anti-tumor activity in vitro of the different formulations loaded with ¹31I-bcl-2ASON or ¹31I-bcl-2/bcl-xlASON or bcl-2ASON or bcl-2/bcl-xlASON. Immunocytohistochemical staining and HE staining were used for in vivo observation, respectively. (6) The ability of NA to image breast cancer in tumor-bearing rats was determined using emission computed tomography (ECT). (7) Results are presented as mean ± standard deviation (SD). Statistical analysis was performed using the ANOVA and t-test with p<0.05 considered as statistically significant difference. RESULTS (1) The conditions of reaction (30min at 60°C, pH5.0; 45min at 60°C, pH5.0; 60min at 60°C, pH7.0 ) currently used gave the better results.The radiolabeling efficiency, radiochemistry purity and specific radioactivity of 125I-ON were 71.66 + 7.73%, 98.33±0.39%, 4.09±0.11 MBq-nmol"1. 125I-ON remained stable in 0.01M HEPES buffer and human serum even after incubation for 4h. (2) Liposomes composed of PC: CH:DSPE-PEG2ooo: DPPG, PC:CH:DSPE-PEG2000: DDAB, PC: CH:DSPE-PEG2Ooo and PC: CH at the molar ratios of 0.50:0.35:0.06:0.10, 0.50:0.29:0.06:0.12, 0.50:0.35:0.06 and 0.50:0.35 respectively, prepared at lOmg/ml in iso-propyl ether-chloroform (l:l,v/v) and chloroform-methanol (2:1, v/v) using a rotary evaporator at 41-42 C and rotary speed at 200rpm were of small size with satisfying polydispersity (PDI) and zeta potential. (3) LCL formulations before extrusion were 504 + 31.76nm in mean diameter with a PDI of 0.107 + 0.008 <, While the mean diameter of all liposomes after extrusion werell5nm+8.5nm with a PDI of 0.103 + 0.002. The zeta potential of NA and PA were -29.23 + 0.45 mV and +41.91+0.58 mV, respectively. The liposomal formulations prepared for thisstudy consisted of 70.28±1.84% and 71.57±2.21% encapsulation efficiency in cases of NA and PA, and 41.66±2.03% in case of A-D and 34.33±3.17% in case of CA (p<0.05) respectively. (4) Compared with PA ^ A-Dn CAand FA, the enhanced cellular uptake (32.51±1.44%) by MCF-7 cells and biological effects were observed in the group of NA (p<0.05) .The nuclear condensation, fragmentation and apoptotic bodies in MCF-7 cells were typical of apoptotic cell death induced by NA-mediated antisense and internal radiation. Corresponding to results of HE staining, the findings of MTT and FCM confirmed the inhibitory effect of NA on MCF-7 cells characterized by decreased cell survial rate and bcl-2 protein level. The inhibitory effect of bcl-2/bcl-xl bispecific ASON was more statistically significant than that of bcl-2-ASON. (5) The four kinds of liposomes and FA under study exhibited bicompartmental behavior regarding clearance. Pharmacokinetic parameters after iv administration demonstrated that both FA and CA were cleared rapidly. NA demonstrated a favourable pharmacokinetic properties in the blood compartment, the TU2 a of 22.11±1.16min, 7V2 P of 393.33±28.58min and area under the concentration-time curve (AUC) of 4119.66±46.96(p<0.05) . As showed by the distribution data in normal mice, spleen and liver were the main organs of accumulation of liposmes. The liver took up NA 30% less than CA (p<0.05) . However, no significant difference in splenic uptake for the four kinds of liposomes because of their same size(p>0.05 ) . The tissue distribution results in normal mice and tumor-bearing rats indicated a large advantage for the anionic-LCL in terms of tumors exposure to the drug and provide a solid rational for the claims of improved therapeutic activity. (6) The percent accumulation of injected dose of NA-D in breast cancer tissues (6.23±0.23%ID/g), the ratios of tumor to blood and tumor to muscle slowly maximized at lOh post injection, most probably due to the "enhanced permeability and retention" (EPR) effect. Hence in radionuclide antisense scintigraphy, the breast cancer of rats was clearly displayed at lOh after iv administration of NA-D.However, tumors were notvisualized in rats with the iv injection of NS and NN even at the delayed time. (7) In the therapeutic studies carried out in tumor-bearing models, NA-D exhibited the most effective anti-tumor activity among all groups under study. When the radioactivity of 131I increased from O.lmCi to 0.2mCi, NA-D achieved the longest tumor growth-inhibiting time (18.3±1.8d) , the highest tumor-growth inhibiting rate (64.53±2.54%), the lowest bcl-2 expression in tumor (1.2±0.2), and the smallest tumor volume among all groups studied after four times iv injection with endurable side effects (p<0.001 ) CONCLUSION Due to the inhibition of rapid uptake of LCL by the reticulo-endothelial system (RES), LCL displays valuable pharmacologic properties characterized by long half-life, slow clearance and large AUC. The inhibitory effect exhibited by NA in this study indicates that NA does improve the tumor cell uptake and drug delivery to both MCF-7 cells and animal models through the passive targeting. What's more, it was the combination of ASON and radiation therapy that contributed to the enhancement of anti-tumor activity. Our data also suggest that the use of bcl-2/bcl-xl bispecific ASON, which most efficiently inhibited bcl-2 expression and induced apoptosis of breast cancer cells, deserves attention as a novel approach to breast cancer therapy. This therapeutic approach also stresses the potential of these anionic liposomal system to enhance anticancer drug delivery to other maglinant tumors.