Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Braunschweig, Germany; Center of Pharmaceutical Engineering, Technische Universität Braunschweig, Braunschweig, Germany.
Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Braunschweig, Germany; Center of Pharmaceutical Engineering, Technische Universität Braunschweig, Braunschweig, Germany.
Eur J Pharm Biopharm. 2018 May;126:115-122. doi: 10.1016/j.ejpb.2017.08.009. Epub 2017 Sep 1.
Due to the limited chemical stability of the natural hyperforin molecule, a more stable form of hyperforin, i.e., the hyperforin dicyclohexylammonium salt (HYP-DCHA) has been used for ex vivo and in vitro experiments in recent years, but its actual stability under typical cell culture conditions has never been studied before. In this contribution the stability of HYP-DCHA was examined under typical cell culture conditions. Different cell culture media with and without fetal calf serum (FCS) supplementation were studied with regard to further stabilization of HYP-DCHA determined with HPLC analysis. Furthermore, albumin nanoparticles were examined as a stabilizing carrier system for HYP-DCHA. In this context, the interaction between HYP-DCHA and albumin nanoparticles (ANP) was examined with regard to size and loading with HYP . The effects of HYP-DCHA either supplied in cell culture medium or loaded on ANP on viability and cytotoxicity were studied in vitro on HaCaT monolayers (human keratinocyte cell line). HYP-DCHA supplied in FCS-containing medium was recovered completely after 24h of incubation. However, a lack of FCS caused a total loss of HYP-DCHA after less than 24h incubation time. Supplying HYP-DCHA loaded on ANP in an FCS-free medium resulted in a recovery of about 60% after 24h incubation. HYP-DCHA supplied in medium along with FCS showed a slow dose-dependent decrease in viability of HaCaT cells without any cytotoxic effects (antiproliferative effect). Treatment with HYP-DCHA with a lack of FCS resulted in a significantly faster decrease in viability which was mainly due to cytotoxicity. The latter was true for HYP-DHCA-loaded ANP where increased cytotoxicity was observed despite the presence of FCS. The results show that the stability of the widely used HYP-DCHA is rather limited under cell culture conditions. Especially a lack of FCS leads to degradation and/or oxidation of HYP-DCHA probably causing an increased cytotoxicity. In contrast, FCS supplementation fairly stabilizes HYP-DCHA under cell culture conditions while albumin nanoparticles may serve the same stabilization purpose despite increasing cytotoxic effects onto the cells themselves.
由于天然金丝桃素分子的化学稳定性有限，近年来，一种更稳定的金丝桃素形式，即金丝桃素二环己基铵盐（HYP-DCHA）已用于体外和体内实验，但在典型的细胞培养条件下，其实际稳定性从未被研究过。在本研究中，研究了典型细胞培养条件下 HYP-DCHA 的稳定性。研究了含有和不含有胎牛血清（FCS）的不同细胞培养基，通过 HPLC 分析确定 HYP-DCHA 的进一步稳定情况。此外，还研究了白蛋白纳米粒作为 HYP-DCHA 的稳定载体系统。在这方面，研究了 HYP-DCHA 与白蛋白纳米粒（ANP）之间的相互作用，包括大小和 HYP 的载药量。在 HaCaT 单层（人角质形成细胞系）上研究了在细胞培养培养基中提供 HYP-DCHA 或负载在 ANP 上的 HYP-DCHA 对细胞活力和细胞毒性的影响。在含有 FCS 的培养基中提供的 HYP-DCHA 在孵育 24h 后完全回收。然而，在没有 FCS 的情况下，孵育不到 24h 后 HYP-DCHA 完全丢失。在不含 FCS 的培养基中提供负载在 ANP 上的 HYP-DCHA 孵育 24h 后可回收约 60%。在含有 FCS 的培养基中提供的 HYP-DCHA 表现出缓慢的剂量依赖性 HaCaT 细胞活力下降，没有任何细胞毒性作用（抗增殖作用）。在没有 FCS 的情况下，HYP-DCHA 的处理导致细胞活力明显更快下降，这主要是由于细胞毒性。对于负载 HYP-DCHA 的 ANP 也是如此，尽管存在 FCS，但观察到细胞毒性增加。结果表明，在细胞培养条件下，广泛使用的 HYP-DCHA 的稳定性相当有限。特别是缺乏 FCS 会导致 HYP-DCHA 降解和/或氧化，可能导致细胞毒性增加。相比之下，在细胞培养条件下，FCS 补充剂相当稳定 HYP-DCHA，而白蛋白纳米粒可能具有相同的稳定作用，尽管对细胞本身有增加的细胞毒性作用。
