Estrone-Targeted Liposomes For Mitoxantrone Delivery Via Estrogen Receptor–Synthesis,characterizations And Antitumor Activities

The overexpression of Estrogen receptor?ER?is widely found in human tumors,thus ER can be a potential target for cancer therapy.To target tumor cells and improve the therapeutic effect on leukemia,a novel mitoxantrone?MTO?formulation,the MTO-loaded estrogen receptor targeted and sterically stabilized liposome?ES-SSL-MTO?,was prepared and characterized.Firstly,estrone?ES?as the ligand of ER was successfully coupled to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino?polyethylene glycol?-2000]?DSPE-PEG2000-NH₂?in a two-step reaction.The intermediate product?ES-COOH?and end product?DSPE-PEG2000-ES?were confirmed using 1H-NMR and matrix-assisted laser desorption/ionization time of flight mass spectrometry?MALDI-TOF?,respectively.Secondly,MTO-loaded liposomes were prepared by the ammonium sulfate gradient method.To optimize the preparation of MTO-loaded liposomes,different conditions were investigated,including the ratio of drug and lipid,volume of ammonium sulfate solution,incubation time,and incubation temperature.Further,the successfully prepared MTO-loaded liposomes were characterized.After separation from the free MTO via dialysis,the drug encapsulated in liposomes was analyzed by UV-visible spectrophotometer at 665 nm to determine the entrapment efficiency?EE?and the drug loading capacity?DL?.Particle size,size distribution,polydispersity index?PDI?and Zeta potential were detected by the dynamic light scanning.The morphological examination on formulation was performed by transmission electron microscopy?TEM?.To explore the stability of formulations,the MTO leaked from liposomes was detected following the storage at 4 °C or 25 °C at different time points.The MTO-loaded liposomes were incubated within fetal bovine serum?FBS?at 37 °C for 24 h to investigate drug release.Later,targeted delivery of estrogen receptor targeted and sterically stabilized liposome?ES-SSL?in vitro and in vivo was studied.To compare cellular uptake of ES-SSL and sterically stabilized liposome?SSL?,HL-60 cells were incubated with coumarin-6?a lipophilic fluorescent probe,entrapped inside the bilayers of liposomes?loaded ES-SSL or SSL for 4 h.The number of fluorescence-positive cells was determined by flow cytometry.Internalization mechanisms of ES-SSL were explored by adding different inhibitory agents of endocytosis to cells before incubating with ES-SSL or SSL.To investigate the distribution of ES-SSL in mice xenografted with HL-60 cells,DiR loaded ES-SSL and SSL were injected intravenously into mice,respectively.At different time points after injection,fluorescence distribution in mice was detected by IVIS spectrum imaging system.At last,the antitumor activity of ES-SSL-MTO was confirmed in vitro and in vivo.The anti-proliferative effect of MTO and various MTO-loaded liposomes on HL-60 cells was measured with CCK-8 kits.Inhibitory effect on tumor growth was evaluated in mice xenografted with HL-60 cells.Results showed that,the chemical structures of DSPE-PEG2000-NH2 and DSPE-PEG2000-ES were confirmed by 1H-NMR spectra.MALDI-TOF MS spectra determined the molecular weights?MW?of DSPE-PEG2000-NH2 as 2175 Da.The MW of DSPE-PEG2000-ES was 2521 Da,which is similar to the theoretical value.The yields of these two reactions were increased to 98% and 72%,respectively.The preparation of MTO-loaded liposomes was optimised using a molar ratio of drug to lipids as 1:15 with 20 mL ammonium sulfate solution under 2 h incubation at 37°C.The dynamic light scanning and TEM results showed that,the size of particles was about 140 nm and homogeneous?PDI=0.12±0.008?,which was appropriate for intravenous administration.The Zeta potential of ES-SSL-MTO was 1.54±0.53 mV.Results specified an obvious advantage of ES-SSL-MTO and SSL-MTO over other liposomes.Following 48 h storage,the drug leakage of ES-SSL-MTO was still less than 8%.In cellular uptake study,the number of fluorescence-positive cells treated with coumarin-6 loaded ES-SSL was 17-fold higher than that of coumarin-6 loaded SSL.This result suggested that the internalization of targeted formulations was achieved in a receptor-dependent manner.Results of internalization mechanisms presented that the internalization of ES-SSL in HL-60 cells was significantly reduced after using sucrose?an inhibitory agent for clathrin-mediated endocytosis?,genistein?an inhibitory agent for caveolae-mediated endocytosis?,and amiloride?an inhibitory agent for macropinocytosis?.It indicated that ES-SSL also induced non-specific or receptor-independent uptake.Results of targeting study in vivo showed that compared to concentration in liver of SSL,the maximum concentration of ES-SSL was distributed in tumor tissues at 6 h.Until 24 h after injection,fluorescence was detected in the tumor tissue.ES-SSL-MTO significantly reduced the cell growth relative to other MTO formulations.IC50 of ES-SSL-MTO was significantly lower than those of L-MTO or SSL-MTO.These results indicated that the cytotoxicity of ES-SSL-MTO was depended on the binding ability of ER.In the in vivo antitumor study,ES-SSL-MTO effectively inhibited the growth of tumor in mice.The tumor inhibition rate of ES-SSL-MTO?86.03%?was significantly higher than those of other MTO formulations.In conclusion,a novel MTO loaded sterically stabilized liposome modified with ES formulation was developed,which showed high encapsulation efficiency and excellent physicochemical characterizations.ES-SSL-MTO was able to significantly improve the targeted delivery of MTO to ER overexpressing tumor cells,and furthermore efficiently inhibit cancer cell growth.In the future,ES-SSL-MTO has a potential to achieve clinical practice.