Guthrie Jeffrey W, Ryu Jang-Ha, Le X Chris, Wiebe Leonard I
Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry and School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
J Pharm Pharm Sci. 2007;10(2):246-55.
Cyclodextrins (CDs) have been identified as a viable alternative to viral vectors for use in therapeutic applications. Here, the stability of the complex formed between the a multiply charged, cationic, fully substituted heptakis-(6-amino-2-galactosyl)cyclodextrin (BCDX12) with a multiply charged 12-mer hexachlorofluorescein tagged arabinopolynucleotide (Hex-PAH) have been evaluated.
The stability of complexes of Hex-PAH and BCD-X12 was studied with respect to mole ratio (1:1, 1:2, and 1:5 Hex-PAH:BCD-X12), pH, buffer concentration, temperature, and agitation using capillary electrophoresis with laser induced fluorescence detection (CE/LIF). Two neutral CDs and an additional cationic CD were also tested under the same analytical conditions to determine their ability to form complexes.
Hex-PAH:BCDX12 complexes at mole ratios of 1:2 were stable in 10 mM (160 mM total borate concentration) sodium tetraborate buffer at pH 7.5 and at temperatures of 4 degrees C and 25 degrees C over 48 hours. However, the Hex-PAH:BCD-X12 complex was less stable at 37 degrees C and at higher buffer concentrations and pH values. Strong vortex mixing prior to analysis was found to disrupt the complex. Of the four CDs tested for their ability to complex with Hex-PAH, only BCDX12 formed stable complexes with Hex-PAH under the test conditions.
Capillary electrophoresis was found to be well suited to test the stability of cyclodextrin-nucleotide complexes. CE/LIF indicated that only a single Hex- PAH:BCD-X12 complex was formed at all formulation ratios, and that the complexes were electrophoretically identical to each other, and increasing the molar ratio beyond 1:2 did not contribute measurably to complex stability. Storage temperature and agitation conditions were found to influence complex stability. Since no stable complexes were formed with neutral cyclodextrins, the results support the hypothesis of a 'charge associated' complex rather than an inclusion complex, although inclusion complexes cannot be excluded on the basis of these studies.
环糊精（CDs）已被确定为用于治疗应用的病毒载体的可行替代品。在此，已评估了多电荷阳离子全取代七聚（6-氨基-2-半乳糖基）环糊精（BCDX12）与多电荷12聚体六氯荧光素标记阿拉伯聚核苷酸（Hex-PAH）形成的复合物的稳定性。
使用激光诱导荧光检测毛细管电泳（CE/LIF），研究了Hex-PAH与BCD-X12复合物在摩尔比（1:1、1:2和1:5 Hex-PAH:BCD-X12）、pH、缓冲液浓度、温度和搅拌条件下的稳定性。还在相同分析条件下测试了两种中性CD和另一种阳离子CD形成复合物的能力。
摩尔比为1:2的Hex-PAH:BCDX12复合物在pH 7.5、10 mM（总硼酸盐浓度160 mM）的四硼酸钠缓冲液中，于4℃和25℃下48小时内保持稳定。然而，Hex-PAH:BCD-X12复合物在37℃以及更高的缓冲液浓度和pH值下稳定性较差。发现分析前强烈涡旋混合会破坏复合物。在测试的四种与Hex-PAH形成复合物能力的CD中，在测试条件下只有BCDX12与Hex-PAH形成了稳定的复合物。
发现毛细管电泳非常适合测试环糊精-核苷酸复合物的稳定性。CE/LIF表明，在所有配方比例下仅形成了单一的Hex-PAH:BCD-X12复合物，且这些复合物在电泳上彼此相同，将摩尔比增加到超过1:2对复合物稳定性没有显著贡献。发现储存温度和搅拌条件会影响复合物稳定性。由于中性环糊精未形成稳定的复合物，尽管基于这些研究不能排除包合物，但结果支持“电荷相关”复合物而非包合物的假设。
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