pH-Responsive Artemisinin Derivatives and Lipid Nanoparticle Formulations Inhibit Growth of Breast Cancer Cells <em>In Vitro</em> and Induce Down-Regulation of HER Family Members

Artemisinin (ART) dimers show potent anti-proliferative activities against breast cancer cells. To facilitate their clinical development, novel pH-responsive artemisinin dimers were synthesized for liposomal nanoparticle formulations. A new ART dimer was designed to become increasingly water-soluble as pH declines. The new artemisinin dimer piperazine derivatives (ADPs) remained tightly associated with liposomal nanoparticles (NPs) at neutral pH but were efficiently released at acidic pH's that are known to exist within solid tumors and organelles such as endosomes and lysosomes. ADPs incorporated into nanoparticles down regulated the anti-apoptotic protein, survivin, and cyclin D1 when incubated at low concentrations with breast cancer cell lines. We demonstrate for the first time, for any ART derivative, that ADP NPs can down regulate the oncogenic protein HER2, and its counterpart, HER3 in a HER2+ cell line. We also show that the wild type epidermal growth factor receptor (EGFR or HER1) declines in a triple negative breast cancer (TNBC) cell line in response to ADP NPs. The declines in these proteins are achieved at concentrations of NP109 at or below 1 µM. Furthermore, the new artemisinin derivatives showed improved cell-proliferation inhibition effects compared to known dimer derivatives.青蒿素（Artemisinin, ART）二聚体对乳腺癌细胞展现出强效抗增殖活性。为推动其临床开发，本研究合成了可用于脂质体纳米制剂的新型pH响应型青蒿素二聚体。所设计的新型青蒿素二聚体可随pH值降低而逐步提升水溶性。该新型青蒿素二聚体哌嗪衍生物（ADPs）在中性pH条件下可与脂质体纳米颗粒（liposomal nanoparticles, NPs）紧密结合，而在实体瘤及内体、溶酶体等细胞器的典型酸性pH环境中可被高效释放。当以低浓度与乳腺癌细胞系共孵育时，负载于纳米颗粒的ADPs可下调抗凋亡蛋白生存素（survivin）与细胞周期蛋白D1（cyclin D1）的表达水平。本研究首次针对所有青蒿素衍生物证实，ADPs纳米颗粒可在HER2阳性细胞系中下调致癌蛋白人表皮生长因子受体2（HER2）及其同源蛋白人表皮生长因子受体3（HER3）的表达；同时发现，在三阴性乳腺癌（TNBC）细胞系中，ADPs纳米颗粒可诱导野生型表皮生长因子受体（EGFR或HER1）的表达水平下降。上述蛋白表达下调均可在浓度≤1 µM的NP109中实现。此外，相较于已知的二聚体衍生物，该新型青蒿素衍生物展现出更优异的抗细胞增殖抑制活性。