School of Pharmacy, Faculty of Medical and Health Sciences, University of Auckland, New Zealand.
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand.
J Control Release. 2019 Jul 10;305:89-100. doi: 10.1016/j.jconrel.2019.05.018. Epub 2019 May 13.
Chemoresistance poses a major challenge in cancer treatment. This study aims to investigate whether intracellular drug delivery using hyaluronic acid (HA) functionalised pH-sensitive liposomes (HA-pSL) can circumvent gemcitabine resistance in pancreatic cancer (PC). HA-pSL were obtained by covalently conjugating HA with preformed pSL. A resistant PC cell line Gr2000 was developed by exposing MIA PaCa-2 cells to gemcitabine, and characterised for their expression of CD44, a receptor for HA, and drug transporters. Cellular uptake and intracellular trafficking of liposomes were determined by confocal microscopy and HPLC analysis of intracellular drug content. Following a pharmacokinetic study in rats, anti-tumour efficacy was compared between MIA PaCa-2 and Gr2000 xenograft mouse models. HA-pSL with an HA density of 179 μg/μmol had a larger size (152.3 vs 136.3 nm), and higher zeta potential (-46.8 vs -10.5 mV) than pSL. The sensitivity of Gr2000 to gemcitabine reduced 444 times compared to its parental cell line, despite no change to the total drug influx, as drug influx- and efflux-transporters in Gr2000 cells were simultaneously up-regulated. Both cell lines had high expression of CD44. HA facilitated cell uptake without compromising the endosome-escape ability of pSL as evidenced by confocal images and co-localization analysis of the dual-fluorescence labelled liposomes and Lysotracker. HA-pSL significantly outperformed pSL, and increased cellular drug influx by 3.6 times in MIA PaCa-2 cells, and 4.6 times in Gr2000 cells. Both liposomes improved the pharmacokinetic profile of free drug. HA-pSL treatment was superior to pSL, and resulted in 6.4 times smaller tumours (weight) in the MIA PaCa-2 xenograft models, and 3.1 smaller in the Gr2000 models compared with the free drug. Taken together, this study highlighted the use of intracellular delivery strategies (HA-CD44 interaction and endosome escape) to overcome gemcitabine resistance, however, the overall improvement was marginal and tumours still existed. Further improvement in delivery efficiency of HA-pSL to target tumours and additional manipulation of the cellular metabolism of gemcitabine are needed to tackle chemoresistance.
化疗耐药性是癌症治疗的主要挑战。本研究旨在探讨使用透明质酸（HA）功能化 pH 敏感脂质体（HA-pSL）进行细胞内药物递送是否可以克服胰腺癌细胞（PC）对吉西他滨的耐药性。HA-pSL 通过将 HA 与预先形成的 pSL 共价偶联获得。通过用吉西他滨暴露 MIA PaCa-2 细胞来开发耐药性 PC 细胞系 Gr2000，并对其 CD44 表达进行特征分析，CD44 是 HA 的受体，以及药物转运体。通过共聚焦显微镜和细胞内药物含量的 HPLC 分析来确定脂质体的细胞摄取和细胞内转运。在大鼠药代动力学研究之后，比较了 MIA PaCa-2 和 Gr2000 异种移植小鼠模型之间的抗肿瘤疗效。HA-pSL 的 HA 密度为 179μg/μmol，其粒径（152.3nm 比 136.3nm）更大，zeta 电位（-46.8mV 比-10.5mV）更高。尽管总药物流入量没有变化，但 Gr2000 对吉西他滨的敏感性比其亲本细胞系降低了 444 倍，因为 Gr2000 细胞中的药物流入和流出转运体同时上调。两种细胞系均高表达 CD44。HA 促进细胞摄取，而不会损害 pSL 的内涵体逃逸能力，这可以通过共聚焦图像和双荧光标记脂质体和 Lysotracker 的共定位分析来证明。HA-pSL 明显优于 pSL，使 MIA PaCa-2 细胞中的细胞内药物流入增加了 3.6 倍，Gr2000 细胞中的药物流入增加了 4.6 倍。两种脂质体均改善了游离药物的药代动力学特征。HA-pSL 治疗优于 pSL，与游离药物相比，MIA PaCa-2 异种移植模型中的肿瘤（重量）小了 6.4 倍，Gr2000 模型中的肿瘤小了 3.1 倍。总之，本研究强调了使用细胞内递药策略（HA-CD44 相互作用和内涵体逃逸）来克服吉西他滨耐药性，然而，总体改善幅度较小，肿瘤仍然存在。需要进一步提高 HA-pSL 对靶肿瘤的递药效率，并对吉西他滨的细胞代谢进行额外的操作，以解决化疗耐药性问题。
