雷公藤红素（Celastrol）是一种从传统中药植物雷公藤（Tripterygium wilfordii）中提取的五环三萜类化合物，在抗炎、抗氧化和抗肿瘤等方面均有疗效，通过多重机制发挥抗肿瘤作用。然而，雷公藤红素的水溶性差和全身毒性问题严重限制了其临床应用，使其难以实现理想的治疗效果。

为解决这一难题，我们创新性地设计了一种可原位注射的温度/pH双响应雷公藤红素衍生物（FCPC），该系统结合了温度敏感性和pH响应性双重特性，实现了药物的精准递送和可控释放。该系统在注入肿瘤组织后，能够在一分钟内迅速形成稳定的凝胶，并在五天内持续释放药物，显著提高了药物在病灶部位的滞留时间。

使用化学合成的方法合成了与雷公藤红素活性相当的哌嗪化雷公藤红素（PIPCel），选用泊洛沙姆F127作为温敏凝胶，并在二者之间引入了pH敏感的2，5-二羟基-4-甲基-2，5-二氧代-3-呋喃丙酸（CDM）。紫外光谱、红外光谱和核磁被用于验证FCPC合成。使用扫描电子显微镜、透射电子系显微镜和原子力显微镜分析FCPC水凝胶的微观结构，并用流变实验表征水凝胶的特性。为了观察4T1细胞对FCPC中的PIPCel的摄取能力，我们合成了香豆素标记的PIPCel及FCPC，在酸性和非酸性培养基中对4T1细胞进行孵育，最后使用共聚焦荧光显微镜观察其对香豆素标记的PIPCel的摄取情况。使用CCK-8、活死染色和流式实验观察FCPC对4T1细胞的抑制与细胞凋亡情况。为了在体内验证FCPC的抑瘤效果和抗复发及转移能力，建立了小鼠原发肿瘤及肺转移模型。最后，使用RNA测序与WB探究了FCPC的抗肿瘤机制。

实验结果表明，我们成功合成了具有规则片状结构的雷公藤红素衍生物FCPC，这一系统在形成过程中展现出了独特的温敏性、可注射性和对肿瘤微环境酸性的响应特性。通过体外实验对FCPC的抗癌效果进行了评估。实验结果显示，在酸性培养条件下，用FCPC处理的4T1乳腺癌细胞的抑制率和凋亡率相较于非酸性培养基中均有显著提升。这一现象表明FCPC在酸性环境中能够更有效地发挥作用，抑制肿瘤细胞生长。此外，细胞所摄取的PIPCel含量显著增多，这一发现进一步证实了FCPC的酸响应特性，即其在酸性环境中能够有效地释放活性成分。

在荷瘤小鼠模型中，我们对FCPC的体内抑瘤效果进行了验证。实验结果显示，FCPC能够显著抑制肿瘤生长，降低肿瘤体积。更重要的是，FCPC还能够降低PIPCel的毒副作用，减少对正常组织的损害。此外，FCPC在抑制肿瘤生长的同时，还能有效抑制肿瘤的复发和肺转移，显示出其在肿瘤治疗中的多重功效。机制研究发现，FCPC可能通过抑制PI3K-Akt和MAPK信号通路来诱导肿瘤细胞坏死和凋亡。PI3K-Akt信号通路在肿瘤细胞的增殖、存活和转移过程中发挥着关键作用，而MAPK信号通路则与细胞的生长、分化和凋亡密切相关。通过抑制这两条信号通路，FCPC能够有效抑制肿瘤细胞的生长和扩散，从而发挥其抗癌作用。

这种创新的注射性温度/pH双响应系统巧妙地解决了雷公藤红素溶解度低、生物利用度差和系统毒性高等问题，为乳腺癌的局部治疗提供了新的策略，展现出良好的临床应用前景，也为开发其他天然抗肿瘤药物的新型递送系统提供了重要参考。

Celastrol, a pentacyclic triterpenoid derived from the traditional Chinese medicinal plant Tripterygium wilfordii, has shown efficacy in anti-inflammatory, antioxidant, and anti-tumor activities, functioning through multiple mechanisms. However, its clinical use is hampered by poor water solubility and systemic toxicity, which hinder its therapeutic potential.

To overcome these limitations, we developed an innovative in-situ injectable temperature/pH dual-responsive celastrol derivative (FCPC). This system integrates temperature sensitivity and pH responsiveness, allowing for precise drug delivery and controlled release. Upon injection into tumor tissue, FCPC rapidly forms a stable gel within one minute and sustains drug release over five days, significantly enhancing drug retention at the tumor site.

We chemically synthesized PIPCel, which has stronger tumor cell inhibition than celastrol, and incorporated it into the thermosensitive gel poloxamer F127, along with the pH-sensitive 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid (CDM). The synthesis of FCPC was confirmed using ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance. The microstructure of the hydrogel formed by FCPC was examined using scanning electron microscopy, transmission electron microscopy, and atomic force microscopy, while its properties were characterized through rheological experiments. To assess the uptake of PIPCel in FCPC by 4T1 cells, we synthesized coumarin-labeled PIPCel and FCPC, incubated them with 4T1 cells in acidic and non-acidic media, and observed the uptake using confocal fluorescence microscopy. The inhibitory and apoptotic effects of FCPC on 4T1 cells were evaluated using CCK-8, live/dead staining, and flow cytometry. To validate the anti-cancer effects and ability to prevent recurrence and metastasis of FCPC in vivo, we established primary tumor and lung metastasis models in mice. Finally, RNA sequencing and Western blotting were employed to investigate the anti-tumor mechanisms of FCPC.

The experimental results demonstrate the successful synthesis of the celastrol derivative FCPC, which features a regular flake-like structure. This system displayed distinct thermosensitive, injectable, and acid-responsive properties in the tumor microenvironment during its formation. In vitro experiments were conducted to assess FCPC's anticancer efficacy. The findings revealed that under acidic culture conditions, FCPC-treated 4T1 breast cancer cells exhibited significantly higher inhibition and apoptosis rates compared to those in non-acidic medium. This indicates that FCPC operates more efficiently in acidic environments to curb tumor cell proliferation. Moreover, a marked increase in cellular PIPCel uptake was observed, further validating FCPC's acid-responsive behavior—specifically, its capacity to release active components effectively in acidic conditions. 

In tumor-bearing mouse models, the in vivo anticancer performance of FCPC was confirmed. Results showed that FCPC significantly suppressed tumor growth and reduced tumor volume. Notably, FCPC also mitigated PIPCel's toxic side effects, thereby lessening harm to healthy tissues. Beyond inhibiting primary tumor growth, FCPC effectively prevented tumor recurrence and lung metastasis, underscoring its multifunctional therapeutic potential in oncology. Mechanistic investigations suggested that FCPC likely triggers tumor cell necrosis and apoptosis by blocking the PI3K-Akt and MAPK signaling pathways. The PI3K-Akt pathway is pivotal for tumor cell proliferation, survival, and metastasis, while the MAPK pathway regulates cell growth, differentiation, and apoptosis. By disrupting these pathways, FCPC effectively hinders tumor cell expansion and dissemination, thereby delivering its anticancer impact.

This innovative injectable temperature/pH dual-responsive system effectively addresses common challenges associated with natural small-molecule drugs, such as low solubility, poor bioavailability, and high systemic toxicity. It offers a promising new strategy for the local treatment of breast cancer and has significant potential for clinical application. Additionally, it provides valuable insights for developing novel delivery systems for other natural anti-tumor drugs.