胰腺癌恶性程度高、预后差，严重威胁人类健康。吉西他滨（gemcitabine，Gem）是治疗胰腺癌的一线化疗药物，但在体内易被代谢失活导致半衰期较短，且因在体内无选择性，常伴有骨髓抑制、胃肠道毒性等多种不良反应。为解决以上问题，基于纳米递送系统负载 Gem 或 Gem 衍生物的研究不断开展并深入，但至今未见突破性进展，也未见有以 Gem 为基础分子的创新纳米药物问世。这可能由于 Gem 极易溶于水，难以构建为包封率高、载药量高、稳定性好等成药性高的纳米递送系统，此外，负载 Gem 或 Gem 衍生物的纳米递送系统的胞内机制研究尚不够清晰，不能为后续研究提供较好的理论指导。

针对以上问题，前期已完成吉西他滨氨基甲酸十四烷酯（N⁴-tetradecyloxy-carbonyl Gem，tcGem）的设计、合成及结构确证，也完成了负载 tcGem 的脂质体（tcGem-loaded liposomes，LipotcGem）的设计、构建与表征、制备工艺筛选与优化等。本文在前期工作基础上，开展了 LipotcGem 的体外活性、体内药效、药代动力学与组织分布及作用机制研究，以全面评价构建的 LipotcGem。期望该脂质体可延长 Gem 的半衰期、高度靶向肿瘤、实现 Gem 的高效低毒，同时也期望通过作用机制的研究阐明 LipotcGem 的胞内生物学行为，为该脂质体的高效低毒提供科学数据支撑。本文受已完成的作用机制结果的启发，设计并构建了共载 tcGem 与线粒体代谢抑制剂 CPI-613 的脂质体（tcGem and CPI-613 co-loaded liposomes，LipoCG），开展了该共载脂质体的系统表征及体外活性等研究与评价，期望多路径实现 Gem 的高效低毒。

分别采用 MTT 法、流式细胞术等方法，以人源胰腺癌细胞系 AsPC-1 和 PANC-1 为细胞模型的体外活性研究结果显示：LipotcGem 在抗肿瘤活性、诱导肿瘤细胞凋亡、阻滞肿瘤细胞周期、抑制肿瘤细胞增殖、迁移与侵袭等方面均显著或极显著优于阳性对照药 Gem。

分别建立小鼠皮下异种移植瘤模型及原位异种移植瘤模型，在该两种模型开展的药效学研究结果表明：LipotcGem 均显著抑制了肿瘤的生长，抑瘤率分别为 69% 和 90%，显著优于阳性对照药 Gem（49% 和 50%）；显著抑制了肿瘤转移，极显著提高了裸鼠的中位生存期（47 天 vs. 30 天）。

以健康小鼠为模型，药代动力学研究结果显示：LipotcGem 的半衰期及药时曲线下面积分别为阳性对照药 Gem 的 3.5 倍（5.23 h vs. 1.46 h）和 8 倍（441.66 h × μmol/L vs. 55.59 h × μmol/L），显著延长了半衰期。以小鼠皮下异种移植瘤为模型，组织分布研究结果表明：LipotcGem 在肿瘤组织中的药物浓度是阳性对照药 Gem 的 750 倍（3.0 μmol/g vs. 0.004 μmol/g），显示出优异的肿瘤靶向能力；LipotcGem 对心脏、脾脏等器官无损伤，安全性好。

以 AsPC-1 细胞为模型的作用机制研究发现：LipotcGem 通过小窝蛋白介导的内吞途径进入细胞，随后定位在溶酶体，释放 tcGem 并转化为 Gem 发挥抗肿瘤细胞的作用；LipotcGem 能够通过降低抗凋亡蛋白 BCL-2 和 MCL-1 的表达，激活 Bax 和 Caspase 3 介导的凋亡途径，致使肿瘤细胞凋亡；LipotcGem 还显著抑制线粒体功能，增强线粒体活性氧生成，加剧肿瘤细胞的损伤。

采用薄膜水化法构建得到共载脂质体 LipoCG，该脂质体的粒径 132 nm，电位 -47 mV，体外活性显著优于LipotcGem。

Pancreatic cancer is characterized by high malignancy and poor prognosis, posing a severe threat to human health. Gemcitabine (Gem) is a first-line chemotherapy drug for the treatment of metastatic or advanced pancreatic cancer. But it is easily metabolized and inactivated in the body and is often accompanied by various adverse reactions such as myelosuppression and gastrointestinal toxicity. To address the above issues, studies on the loading of Gem or Gem derivatives based on nano-delivery systems have been continuously carried out and deepened. However, no breakthrough progress has been achieved in relevant research so far, and no innovative nanomedicines with Gem as the basic molecule have been introduced to the market. This may be because Gem is extremely soluble in water, making it difficult to construct a nano-delivery system with high encapsulation efficiency and long-term stability. In addition, the intracellular mechanism of the nano-delivery system loaded with Gem or Gem derivatives has not been clearly studied, thus cannot provide theoretical guidance for subsequent research.

In view of the above, a novel Gem derivative (N⁴-tetradecyloxycarbonyl gemcitabine, tcGem) has been synthesized and encapsulated into liposomes (LipotcGem) to overcome the above shortcomings in our previous work. In this study, a comprehensive research and evaluation of the constructed LipotcGem were carried out via in vitro and in vivo pharmacodynamics, pharmacokinetics, tissue distribution, and mechanism of action studies, etc. It is expected that LipotcGem can exhibit high efficacy and low toxicity compared with Gem.

In the in vitro activity study, LipotcGem exhibited significant anti-tumor activity in human pancreatic cancer cell lines AsPC-1 and PANC-1. It significantly induced tumor cell apoptosis, effectively arrested the tumor cell cycle, inhibited tumor proliferation, and remarkably suppressed tumor cell migration and invasion.

In the in vivo efficacy study, LipotcGem significantly inhibited the growth of tumors in both the subcutaneous xenograft model and the orthotopic xenograft model, with tumor inhibition rates reaching 70% and 90%, respectively, compared to Gem (49% and 50%). Additionally, LipotcGem significantly inhibited tumor metastasis and increased the survival rate of nude mice (50 Days vs. 35 Days).

In the pharmacokinetics and tissue distribution studies, the half-life and the area under the plasma concentration-time curve of LipotcGem were 3.5 times (5.23 h vs. 1.46 h) and 8 times (441.66 h × μmol/L vs. 55.59 h × μmol/L) higher than those of free Gem, respectively. After treatment with LipotcGem, the drug concentration in tumor tissues was 750 times (3.0 μmol/g vs. 0.004 μmol/g) higher than that of free Gem, demonstrating the excellent tumor targeting ability. LipotcGem had no significant toxicity to the organs such as the heart and spleen with good safety.

Mechanistic studies using AsPC-1 cells revealed that LipotcGem enters cells via caveolin-mediated endocytosis, localizes to lysosomes, and releases tcGem, which is subsequently converted to Gem to exert antitumor effects. LipotcGem induces tumor cell apoptosis by downregulating anti-apoptotic proteins BCL-2 and MCL-1 and activating Bax and Caspase 3-mediated apoptosis pathways. Additionally, LipotcGem significantly impairs mitochondrial function, enhances mitochondrial reactive oxygen species production, and exacerbates tumor cell damage.

Additionally, inspired by the above mechanistic insights of LipotcGem, a co-loaded liposome with tcGem and the mitochondrial metabolism inhibitor CPI-613 (named LipoCG) was designed and constructed using the thin-film hydration method. LipoCG exhibited a particle size of 132 nm and a zeta potential of -47 mV. The in vitro antitumor activity of LipoCG was significantly superior to that of LipotcGem.