Studies On Active Targeting Liposomes Directed By Chimeric TNT-3 Monoclonal Antibody

Liposomes are composed of phospholipid bilayer structures that encapsulate an aqueous interior. Liposome properties and biological behaviors can be controlled by surface modification. Attachments of hydrophilic polymers, such as poly (ethylene glycol), enable them to have a prolonged circulation time without being opsonized and recognized by mononuclear phagocytic system. Attachments of specific ligands, such as monoclonal antibody or peptides, make liposomes own active targeting effect to specific cells or tissues.Chimeric Tumor Necrosis Therapy -3 monoclonal antibody (chTNT-3) is a novel monoclonal antibody (MAb) which can bind to degenerating cells located in necrotic regions of solid tumors. Since 50-90% of the progeny of tumor cells shortly undergoes degeneration and cell death, chTNT-3 can be utilized to direct at nuclear antigens accessible in necrotic areas of various tumors, while conventional tumor cell MAbs can only target to one kind of tumor cell antigen.Lipid materials for preparing sterically stabilized liposomes, such as methoxypolyethyleneglycol-hydrogenated soy phosphatidyl-ethanol-amine (mPEG-HSPE) and pyridylthiopropionoylamino-PEG-hydrogenated soy phosphatidylethanol-amine (PDP-PEG-HSPE), were synthesized and determined via TLC, IR, ¹H-NMR, etc.Preparation of active targeting liposomes were carried out via two steps: (1). The homogenized sterically stabilized liposomes (PDP-SL) with PDP-PEG-HSPE as lipid material were prepared via high pressure extrusion method. The antitumor agent, doxorubicin was then encapsulated using ammonium sulfate gradient method. According to the indexes of encapsulation efficiency and drug load, via central composite design-response surface methodology, an optimized liposome formulation was obtained as follow: HSPC/CHOL/mPEG-HSPE/PDP-PEG-HSPE = 5:4:0.2:0.05 (molar ratio), drug-lipid ratio=0.18:1 (w/w). The temperature and time for encapsulation were 65℃C and 20 min. The encapsulation efficiency was about (93.2±2.0)%. (2). PDP-SLs were reduced by dithiothreitol (DTT) to form thiol groups on the surface of liposomes (HS-SL). MPB-chTNT-3 were then linked to HS-SLs to form the stabilized immunoliposomes (chTNT-3-SL). Besides this, SAv-SL could be obtained by linking MPB-streptavidin (MPB-SAv) to HS-SL. chTNT-3 could be derivatized to chTNT-3/B with biotin substitute degree between 3-8. Both chTNT-3/Band SAv-SLs composed of pretargeting preparations.After antibody or SAv modification, the average sizes of chTNT-3-SL and SAv-SL were increased from 102 nm (PDP-SLs) to 123 and 114 nm, respectively. The binding efficiencies of antibody or SAv on liposome surfaces were 69.8% and 82.6%. The surface densities of antibodies or SAv proteins were 108.9 and 51.5μg/μmol PL. The immunoreactivity of chTNT-3 derivatives were determined by ELISA, and the results showed that it was well remained. After being conjugated to the surface of liposomes, the immunoreactivity was kept as well to a certain extent. The preliminary stability test indicated that after 14 days of storage at 4℃, the mean sizes and size distributions of both chTNT-3-SL and SAv-SL changed little, the drug leakage was less than 3%, the relative immunoreactivity of chTNT-3-SL kept unchanged and the physical and chemical properties were stable.To verify the in vitro targeting effect of active targeting liposomes, fixed Raji cell models were established and calcein loaded active targeting liposomes were prepared. The specific binding effects of both sterically stabilized liposomes and pretargeting preparations to fixed Raji cells were investigated by fluorescence assay. The results showed that the binding effects of two kinds of liposomes were both higher than those in control groups (P<0.001).Followed two-compartment model, the biological half-life of chTNT-3-SL[DXR] and (chTNT-3/B + SAv-SL[DXR]) in rats was longer than that of free doxorubicin but shorter than sterically stabilized liposomes. ChTNT-3-SL[DXR] could target to the necrosis site of tumor and its accumulation in tumor was a gradual process. Pharmacokinetic parameters of rats after iv DXR and DXR preparations were as follow: t_1/2 of SL[DXR], chTNT-3-SL[DXR] and (chTNT-3/B + SAv-SL[DXR]) were 21.9h, 21.6h and 15.0h, respectively; AUC0.72 of them were 2241μg·h/mL, 1440μg·h/mL and 1138μg·/mL, respectively; MRT_0-72 of them were 20.1 h, 16.9h and 13.8h, respectively. In biodistribution investigation of mice bearing H460 tumor, the DXR concentration of pretargeting[DXR] group was the highest in all groups at 4h and 24h and was significantly higher than SL[DXR] and free DXR groups at 4h (P<0.05). And at 48h the DXR concentration of chTNT-3-SL[DXR] group was highest in all groups. The advantages of antitumor efficacy of chTNT-3-SL[DXR] and chTNT-3/B+Strepavidin-SL[DXR] pretargeting preparations to subcutaneous transplantation tumor varied. ChTNT-3-SL[DXR] had a better antitumor activity with a final tumor inhibitory rate of 59.5%, while the pretargeting preparations had thehighest antitumor efficacy with tumor inhibitory rate of 61.9% during some intervals, which was better than that of SL[DXR]'s (49.1%).