Studies on preparation of HER-2 targeting boron liposomes and the influencing factors

Objective To develop HER-2 targeting boron-containing liposomes as the potential drug delivery vehicle for boron neutron capture therapy, studies on optimization of the developing conditions and targeting specificity of the prepared liposomes were carried out. Methods The liposomes were prepared by freezing-thawing and extrusion, and the loading of boron was performed using a pH gradient. Several drug-to-lipid ratios were tested for suitable entrapment efficiency. The effects of conjugation time (1 h or 3 h) on the yield of Trastuzumab conjugated to the micellar lipids DSPE-PEG-NHS were tested. Using the micelle transfer method, incorporation of Trastuzumab-PEG-DSPE into liposomes was studied after different temperature and time. The stability of the conjugate was tested by incubation of the liposomes diluted in the culture medium for up to 2 weeks at 4℃ or 37℃.To analyze the effect of temperature during incorporation, Trastuzumab was incubated for 4 h at 4, 20, 37 and 60℃ , then its receptor binding capacity was tested. The targeting specificity was studied in cultured SK-BR-3 cells by increasing amounts of free Trastuzumab in the medium. Results Examined under cryo-transmission microscopy, the prepared liposomes were about 100 nm in diameter, and boron crystals could be seen in the middle. The entrapment efficiency for the tested drug-to-lipid ratios was over 95%. One hour at room temperature was considered sufficient for the conjugation of Trastuzumab to the micellar lipids DSPE-PEG-NHS. Four hours at 60℃ gave the best yield in the incorporation of Trastuzumab-PEG-DSPE into liposomes. After 2 weeks at 37℃ 95% of the Trastuzumab remained still in the liposome fraction. Binding of Trastuzumab to SK-BR-3 cells was not considerably changed after incubated for 4 h at 4, 20, 37 and 60℃. The liposomes showed specific binding to the HER-2 receptor in SK-BR-3 cells. Conclusions The liposomes we prepared appear to be round, well separated and uniform in size with high entrapment efficiency. The liposomes are stable and keep the capacity of binding specificity.;