Institute for Heath and Wellbeing Research, Robert Gordon University, Aberdeen, AB10 7GJ, UK.
Int J Pharm. 2015 Feb 1;479(1):103-17. doi: 10.1016/j.ijpharm.2014.12.058. Epub 2014 Dec 27.
The biocompatibility and cellular uptake of polymer, insulin polyelectrolyte complexes (PECs) prepared using polyallylamine-based polymers was evaluated in-vitro using Caco-2 cell monolayers as a predictive model for human small intestinal epithelial cells. Poly(allyl amine) (PAA) and Quaternised PAA (QPAA) were thiolated using either carbodiimide mediated conjugation to N-acetylcysteine (NAC) or reaction with 2-iminothiolane hydrochloride yielding their NAC and 4-thiobutylamidine (TBA) conjugates, respectively. The effect of polymer quaternisation and/or thiolation on the IC50 of PAA was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay carried out on Caco-2 cells (with and without a 24 h recovery period after samples were removed). Uptake of PECs by Caco-2 cells was monitored by microscopy using fluorescein isothiocyanate (FITC) labelled insulin and rhodamine-labelled polymers at polymer:insulin ratios (4:5) after 0.5, 1, 2 and 4 h incubation in growth media (±calcium) and following pre-incubation with insulin. MTT results indicated that quaternisation of PAA was associated with an improvement in IC50 values; cells treated with QPAA (0.001-4 mg mL(-1)) showed no signs of toxicity following a 24 h cell recovery period, while thiolation of QPAA resulted in a decrease in the IC50. Cellular uptake studies showed that within 2-4 h, QPAA and QPAA-TBA insulin PECs were taken up intracellularly, with PECs being localised within the perinuclear area of cells. Further investigation showed that uptake of PECs was unaffected when calcium-free media was used, while presaturating insulin receptors affected the uptake of QPAA, insulin PECs, but not QPAA-TBA PECs. The biocompatibility of PAA and uptake of insulin was improved by both thiol and quaternary substitution.
采用 Caco-2 细胞单层作为人小肠上皮细胞的预测模型，评估了使用基于聚烯丙胺的聚合物制备的聚合物、胰岛素聚电解质复合物（PECs）的生物相容性和细胞摄取。通过使用碳二亚胺介导的与 N-乙酰半胱氨酸（NAC）的缀合或与 2-亚氨基硫代盐酸盐的反应，将聚烯丙胺（PAA）和季铵化聚烯丙胺（QPAA）分别巯基化以得到它们的 NAC 和 4-硫丁基脒（TBA）缀合物。通过在 Caco-2 细胞（在去除样品后有或没有 24 小时恢复期）上进行 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴化物（MTT）测定，确定聚合物季铵化和/或巯基化对 PAA 的 IC50 的影响。通过荧光素异硫氰酸酯（FITC）标记的胰岛素和罗丹明标记的聚合物在生长培养基（±钙）中孵育 0.5、1、2 和 4 小时后，通过显微镜监测 Caco-2 细胞对 PEC 的摄取（在与胰岛素预孵育之后）。MTT 结果表明，PAA 的季铵化与 IC50 值的提高有关；用 QPAA（0.001-4 mg mL（-1））处理的细胞在 24 小时细胞恢复后没有显示出毒性迹象，而 QPAA 的巯基化导致 IC50 降低。细胞摄取研究表明，在 2-4 小时内，QPAA 和 QPAA-TBA 胰岛素 PEC 被细胞内摄取，PEC 定位于细胞的核周区。进一步的研究表明，当使用无钙培养基时，PEC 的摄取不受影响，而胰岛素受体的预饱和会影响 QPAA、胰岛素 PEC 的摄取，但不影响 QPAA-TBA PEC 的摄取。巯基和季铵化取代均改善了 PAA 的生物相容性和胰岛素的摄取。
