HER-2靶向硼脂质体的制备与影响因素研究

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

目的制备具有良好靶向性的硼脂质体,为硼中子俘获治疗的研究与应用建立一个有效的靶向给药途径。方法采用新型的主动载药方法pH梯度法制备硼脂质体,分析不同药脂比例对包封率的影响,优化Trastuzumab连接到DSPEPEGNHS分子微团的时间因素,采用微团转移法将TrastuzumabPEGDSPE掺入预先制备的硼脂质体,比较不同时间和温度对掺入量的影响,测试脂质体的稳定性、温度对Trastuzumab与受体结合能力的影响以及硼脂质体的受体靶向特异性。结果制备的硼脂质体大小均匀,直径约为100nm,圆形小囊状,药物包封率高。Trastuzumab与DSPEPEGNHS室温下孵育1h可达到足够高的连接率。60℃的水浴中作用4h,90%的TrastuzumabPEGDSPE掺入了脂质体脂膜。脂质体稳定,37℃保存2周仍有95%的抗体与脂质体相连。实验所用温度对Trastuzumab与HER2结合的能力影响不大。制备的硼脂质体与HER2受体的结合能被浓度不断增高的Trastuzumab所置换。结论本法制备的硼脂质体大小均匀,外观圆整,药物包封率高,有良好稳定性。实验过程对抗体特性无明显影响,硼脂质体具备与HER2受体特异性结合的能力。

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 wet：e 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.