Salmaso Stefano, Bersani Sara, Pennadam Sivanand S, Alexander Cameron, Caliceti Paolo
Pharmaceutical Sciences Department, University of Padua, Via F. Marzolo 5, 35131 Padua, Italy.
Int J Pharm. 2007 Aug 1;340(1-2):20-8. doi: 10.1016/j.ijpharm.2007.03.010. Epub 2007 Mar 12.
A thermoresponsive polymer, N-isopropylacrylamide-co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 degrees C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein-co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85+/-3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.
一种具有37℃低临界溶液温度（LCST）的热响应性聚合物N-异丙基丙烯酰胺-共-丙烯酰胺（Mn 6 kDa）被活化并与抗生物素蛋白偶联，得到分子量为200 kDa的衍生物。凝胶渗透分析表明，新的生物共轭物具有与220 kDa球状蛋白相对应的表观尺寸。光子相关光谱和比浊法研究表明，该生物共轭物经历了温度依赖性的相变。蛋白质-共聚物生物共轭物表现出与原始合成共聚物相同的起始相变温度（LCST）。然而，与原始聚合物相比，生物共轭物在较高温度下的聚集曲线发生了偏移。这表明共聚物从卷曲到球状转变的聚集行为通过锚定在蛋白质表面而发生了改变。圆二色性分析表明，共聚物偶联不会改变蛋白质的三级结构和芳香族氨基酸环境。该生物共轭物对生物素和生物素-单克隆抗体保持了85±3%的天然抗生物素蛋白亲和力，并且在三个加热循环后仍保持高亲和力。药代动力学研究表明，共聚物偶联增加了抗生物素蛋白在血液中的停留时间。抗生物素蛋白-共聚物的分布相比天然蛋白质长20倍。共聚物偶联使抗生物素蛋白的分布范围降低了三倍，并显著减少了其对肝脏的摄取。
