Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: In vitro studies
Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: In vitro studies
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DOI：
10.1021/bc9801124
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发表时间：
1999-03-01
影响因子：
4.7
通讯作者：
Zalipsky, S
中科院分区：
文献类型：
--
作者：
Gabizon, A;Horowitz, AT;Zalipsky, S
以叶酸-聚乙二醇-二硬脂酰磷脂酰乙醇胺（FA-PEG-DSPE）为偶联物，通过马来酰亚胺（马来酰亚胺）介导的偶联反应，合成了分子量为2000和3350 Da的PEG衍生的FA-PEG-DSPE。通过H-1 NMR、MALDI-TOF和HPLC分析羧肽酶G的酶裂解来表征缀合物。作为叶酸受体（FR）靶向系统的原型，在氢化磷脂酰胆碱/胆固醇脂质体中以0.5摩尔%磷脂配制缀合物，有或没有另外的甲氧基PEG 2000-DSPE。用各自含有高和低密度FR的M109（鼠肺癌）和KB（人表皮癌）细胞亚系进行体外结合研究。FA-PEG-DSPE显著增强脂质体与肿瘤细胞的结合。当FA-PEG脂质体不含额外的mPEG-脂质时，观察到最佳结合。事实上，我们的实验表明，在脂质体表面上的mPEG的存在下显着抑制FA-PEG-脂质体结合FR。增加PEG系链的分子量从2000至3350 Da改善FR结合，特别是在mPEG包被的脂质体的情况下。FA-PEG脂质体与M109-HiFR细胞非常紧密地结合，如通过游离FA（在孵育开始或结束时以700倍过量使用）不能阻止细胞结合所证明的。这与mPEG-FA的细胞结合活性比游离FA低5-10倍相反，并且可能与脂质体结合的FA的多价性质有关。通过暴露于pH 3（解离FA-FR的条件），只能去除22%的FA-PEG 3350和32%的FA-PEG 3350/mPEG细胞相关脂质体，这表明超过三分之二的结合脂质体在孵育期间被内化24小时在37摄氏度。FA靶向脂质体还显示出与细胞外组织培养组分的非特异性结合增强，这是在短孵育时间实验中特别相关的现象。
Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by H-1 NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.