Caliceti Paolo, Salmaso Stefano, Semenzato Alessandra, Carofiglio Tommaso, Fornasier Roberto, Fermeglia Maurizio, Ferrone Marco, Pricl Sabrina
Department of Pharmaceutical Sciences, University of Padua, Via F Marzolo 5, 35131 Padua, Italy.
Bioconjug Chem. 2003 Sep-Oct;14(5):899-908. doi: 10.1021/bc034080i.
beta-Cyclodextrin-poly(ethylene glycol)-folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure: (1). synthesis of CD-PEG-NH(2) by reaction of monotosyl-activated beta-cyclodextrin with excess of 700 Da diamino-PEG; (2). synthesis of CD-PEG-FA by reaction of CD-PEG-NH(2) with succinimidyl ester-activated folic acid. The CD-PEG-NH(2) intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH(2) and CD-PEG-FA were analyzed by mass spectrometry, (1)H NMR, and UV-vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates: the CD-PEG-NH(2) intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of beta-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified beta-cyclodextrin (16.3 mM). Phase solubility diagrams of beta-estradiol revealed that drug solubility increases from 11 microM in buffer to 600 microM in the presence of beta-cyclodextrins and 5900 microM with CD-PEG-FA. However, the affinity of beta-estradiol for beta-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with beta-cyclodextrin. Computer modeling studies showed that the folic acid linked to the beta-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified beta-cyclodextrin.
β-环糊精-聚乙二醇-叶酸共轭物（CD-PEG-FA）通过两步法合成：（1）. 通过单对甲苯磺酰基活化的β-环糊精与过量的700 Da二氨基聚乙二醇反应合成CD-PEG-NH₂；（2）. 通过CD-PEG-NH₂与琥珀酰亚胺酯活化的叶酸反应合成CD-PEG-FA。CD-PEG-NH₂中间体通过在丙酮中沉淀进行纯化，CD-PEG-FA则通过凝胶渗透和C-18反相色谱法纯化。CD-PEG-NH₂和CD-PEG-FA均通过质谱、¹H NMR和紫外可见光谱进行分析。所有分析方法均证实了共轭物的理论组成：CD-PEG-NH₂中间体由摩尔比为1:1的CD和PEG组成，而CD-PEG-FA由摩尔比为1:1:1的β-环糊精、PEG和叶酸组成。与未修饰的β-环糊精（16.3 mM）相比，CD-PEG-FA共轭物在缓冲液中具有高溶解性（>42 mM）。β-雌二醇的相溶解度图表明，在β-环糊精存在下，药物溶解度从缓冲液中的11 μM增加到600 μM，而在CD-PEG-FA存在下增加到5900 μM。然而，β-雌二醇与β-环糊精的亲和力随着PEG和叶酸共轭作用降低了约4倍。使用苯丁酸氮芥进行的稳定性研究证实，该共轭物在缓冲液中可部分防止药物降解，尽管这种效果远低于β-环糊精。计算机模拟研究表明，通过PEG间隔基连接到β-环糊精上的叶酸可部分与环糊精腔相互作用。最后，与未修饰的β-环糊精相比，CD-PEG-FA的溶血作用降低。
