微环境调控协同层次化“靶向-释药”策略促中药脂质体肿瘤深层递送研究

Anti-breast cancer lipsomes are capable of reducing cytotoxicity of drug and enhancing tumor-site retention greatly, however, there are still big challenges in curative effect. Based on the our previous research of anticancer drug delivery in tumor, the poor therapy effect was attributed to abnormal tumor microenvironment and consequent failure to deep penetration. Therefore, a sequential "trageting-release" lipsome was developed by hierarchical assembly technology using tanshinone IIA, capable of promoting blood circulation for removing blood stasis, as microenvironment regulator, and celastrol as anticancer component in this study. On the initial stage, such drug delivery system releases tanshinone IIA to modulate microenvironment and improve drug delivery at superficial tumor tissue; on the subsequent stage, it releases celastrol at deep tumor to enhance anticancer effect. In addition, 3D tumor sphere model, intratumoral layer by layer scanning and confocal laser scanning microscopy were used to evaluate the penetration and pharmacodynamics. In summary, we fabricate a “deep and accurate” tumoral delivery lipsome by integrating the superiorities of tanshinone IIA and celastrol under the guidance of traditional Chinese medicine (TCM) theory. It can not only help us to find a new breakthrough in breast cancer treatment, but also provide a novel strategy and technology to smart TCM-lipsome tumor delivery.

抗乳腺癌脂质体具有显著降低包埋药物的毒性、提高瘤内滞留的优势，但在改善患者生存时间上依然存在局限。基于以往对抗肿瘤药物瘤内递送规律深入研究发现，肿瘤微环境异常致使药物难以被递送至肿瘤深层是脂质体疗效不佳的主要原因。针对上述问题，本项目引入活血化瘀中药成分丹参酮IIA作为“微环境调控剂”，以雷公藤红素作为抗肿瘤成分，借助分级装配技术构建一种可层次化“靶向-释药”的脂质体。该递药系统在肿瘤外周一级释放丹参酮IIA修复肿瘤微环境，改善药物递送环境；在肿瘤深层二级释放雷公藤红素精确杀伤肿瘤细胞，提高抗肿瘤疗效。同时，采用3D瘤球模型、瘤内逐层扫描和激光共聚焦等技术评价该脂质体瘤内渗透和抗肿瘤疗效。本项目在中医药理论的指导下，整合活血化瘀中药和抗肿瘤中药的优势，构建了兼具“肿瘤深层渗透”和“肿瘤精确递送”特性的脂质体，为治疗乳腺癌寻求新突破点，也为中药多组分抗肿瘤脂质体智能化递送提供新思路和新方法。

肿瘤微环境异常所致的药物递送障碍是目前脂质体研发面临的最大挑战。本研究以丹参酮IIA磺酸钠作为“微环境调控剂”，以雷公藤红素纳米粒作为靶向抗肿瘤“主力”，构建一种可层次化“靶向-释药”的脂质复合系统。首先采用经典透析法成功制备了叶酸修饰的雷公藤红素微乳（脂质复合系统的核心），然后采用多种磷脂/制备工艺对脂质体（脂质复合系统的外壳）制备工艺进行优化，雷公藤红素微乳和丹参酮ⅡA磺酸钠的脂质复合系统(T/CM-L)最优处方为：以大豆卵磷脂为膜材，以薄膜分散法作为制备方法，包封率最高，形态最圆整，稳定性最佳。逐级释放实验中，我们发现T/CM-L的雷公藤红素和丹参酮IIA磺酸钠8 h累积释放量分别为40.4 ± 2.2 %和87.3 ± 3.8 %。细胞学实验可知，T/CM-L抑制MCF-7细胞增殖作用明显强于裸药组和普通脂质体组；细胞摄取量也显著高于游离组；可诱导59.6 ± 2.3%细胞凋亡；3D瘤球模型体外渗透结果表明，脂质复合系统的核心部分—微乳渗透深度为60 μm，显著高于脂质复合系统组。体内生物分布研究表明，DiD标记的T/CM-L（DiD/T/CM-L）在肿瘤部位聚集能力最高，在肝脾等部位聚集较少，肿瘤靶向性明显优于普通脂质体和物理混合组。体内抗肿瘤药效学和安全性实验表明，T/CM-L对MCF-7荷瘤裸鼠的抑瘤率为71.5%，与裸药组和普通脂质体组相比，抑瘤能力明显增强，且对肝肾等主要正常器官以及血液无明显毒性。体内抗肿瘤增效机制评价发现，T/CM-L治疗后动物肿瘤组织（CD31荧光标记）血管数量和密度显著低于各对照组；瘤组织肿瘤相关成纤维细胞（α-SMA荧光标记）的阳性率为7.2%，显著低于各对照组。此外，我们还构建了金纳米棒锚定的热敏T/CM-L，37℃和42℃下雷公藤红素累计释放速率分别为59.6 ± 3.6%和78.9 ± 5.3%。细胞实验表明，近红外照射后系统对MCF-7细胞的IC50为1.5 ± 0.1 μM，可诱导25%细胞凋亡。总而言之，T/CM-L能够逐级释放相应的药物，具有较高的肿瘤靶向性，可显著提高体内外抗肿瘤疗效，安全性可控，通过金纳米棒修饰后，系统的控释能力得到进一步提高，有望用于乳腺癌联合治疗、增效减毒的后续研究。