针对白血病干细胞和微小残留病灶的序级靶向纳米递药系统

Relapsed or refractory leukemia remains the most common therapeutic problem in clinical treatment. The key therapy of refractory leukemia is to reverse the multidrug resistance of leukemia cells in minimal residual disease and kill the leukemia stem cell in stem cell niche thoroughly. In this program, we present the multi-step targeting nano drug delivery system in which alendronate and folate are used as the stepped targeting ligand of the nanolipid particles. Alendronate, a therapeutic drug for osteoporosis, has the specific combination capacity with bone, so it can mediate the nanocarrier to the bone inner membrane after the intravenous administration. This tissue is very closely related with the leukemia stem cell nich, and also most drug-resistence leukemia cell are concentrated here, so there the nanoparticles are accumulated with high drug amount.By combinating the folate-modifies nanopartilces with the folate receptor expressed on these cell,the nanocarriers would be uptaken by endositisis of the cells in large amount and then released the much more amount molecule-targeting drug in,therefore, the kill effect of leukemia stem cell or MDR cell would be increased significantly.The subject will study the transport process and its mechanism of the multistep targeting nanoparticle from system circulation to bone inner membrane, which has not been known recently.It is expected such a drug delivery can be performed by the stepped targeting ligands. The study try to illustrate it is possible that the increase of the drug distribution on the bone active growth area can be reached by using the alendronate modified nanocarrier and try to understand the relation between the concentrated drug in this area and the elimination effect of leukemia stem cells as well as the MDR leukemia cells. In this program the trans-vitamin A acid is used as the chemotherapy inducer to treat the model mice beared with minimal residul desease of leukemia in order to investigate the effect on cell folate express and the inceased kill efficiency of nanoparticels. All the undergoing work will reveal a novel and promising therapeutic method for the leukemia and relaped leukmia, which may be very interesting to the patients who suffer from the disease.The design of step targeting and the expected results might be beneficial not only to the research of drug delivery also be to the area of leukemia research .

常规诱导化疗对难治性白血病效果差且易复发，与药物很少到达骨腔有关，与骨腔中白血病干细胞受细胞龛保护以及微小残留病灶的耐药性有关。课题构建一种有针对性的序级靶向脂质纳米递药系统。以具有趋骨特异性的阿伦磷酸为第一级靶基，介导纳米粒到达与白血病干细胞（龛）关联的骨内膜高骨转换区，特异性地提高病源区药物浓度；根据白血病干细胞和微小残留病灶叶酸受体过度表达的特征及其与正常造血细胞的差异，以叶酸为第二级配基，增加白血病干细胞和耐药细胞对载体的摄取，减少外排，增强分子靶向药物硼替佐米的有效性，抑制白血病细胞增殖。课题研究纳米粒从骨外膜向骨腔内序级靶向转运的可行性、高骨转换区药浓与白血病干细胞及微小残留病灶清除的关系、叶酸受体诱导剂全反式维甲酸在主动靶向治疗中的作用等。课题针对难治性白血病的序级靶向设计未见报道，对阐明纳米粒在骨-造血系统的转运和探讨白血病复发机制以及制订治疗方案具有重要研究价值和应用价值

化疗后存留于骨髓腔中的白血病干细胞和微小残留病灶是白血病复发的原因，这与药物很少能够到达骨腔有关。本项目选择具有趋骨特异性的阿伦磷酸为第一级靶基，介导纳米粒到达与白血病干细胞（龛）存在的骨内膜高骨转换区，提高病源区药物浓度；根据白血病细胞叶酸受体过度表达的特征，以叶酸为第二级配基，增加肿瘤细胞对载体的摄取，实现药物对骨腔和癌细胞的序级靶向。设计和制备了两类携载白血病治疗药物米托蒽醌（MTO）的序级靶向纳米载体：以脂质及修饰聚乙二醇100硬脂酸脂（S-100）为主的脂质纳米粒以及以改构聚乙二醇-维生素E琥珀酸酯（TPGS）组装的聚合物胶束。用阿伦磷酸（ALN）和叶酸分别修饰S-100，制备了二者共修饰、适合渗透骨腔微细血管的ATO纳米粒。建立弱碱性水合介质增强叶酸亲水性专利技术，解决叶酸因疏水性影响靶向的问题。建立羟基磷灰石-白血病细胞体外吸附-解吸附模型和K562/Cur白血病裸鼠模型。健康鼠药动学表明该载体延长血液循环时间，AUC是普通静脉注射液的63倍，股骨摄取率为4.97倍。体内外模型均证明，ALN修饰增强骨组织吸附但不干扰叶酸与K562细胞叶酸β受体的结合。反维A酸诱导上调叶酸β受体表达的白血病细胞摄取显著增加。增强在白血病模型鼠股骨两端骨骺及脊柱的分布，并且富集在白血病干细胞所处高骨转换区，显著降低骨髓中和外周血中白血病细胞比例至正常水平，而肝脾则处于轻微浸染水平，证明该载体有效杀灭或抑制白血病干细胞和微小残留病灶。为增强对白血病耐药细胞的作用，选用具有抑制多药耐药作用的TPGS替代S-100。用ALN及叶酸分别修饰TPGS，将MTO与TPGS用二硫键连接臂链接，以特定比例将三者自组装成双靶向、肿瘤环境敏感、化学与物理载药结合的高载药量的TPGS衍生物胶束。对该载药胶束的研究证实了前述脂质纳米粒的全部体内外序级靶向结果，并且证明，环境敏感设计发挥了更好的抗肿瘤活性，与非敏感型载体比较，体外肿瘤细胞凋亡率分别为29.5%和9.03%。虽然两者药动学行为相似，但肿瘤和骨靶向效果分别是非靶向载体的1.4和1.1倍，阳性破骨细胞显著减少而凋亡细胞显著增多，更强的抑瘤率（52.30% vs 48.25%）, 延长荷瘤鼠生存期(52d vs 47d)。在序级靶向纳米载体设计、制备新技术、体内外序级靶向研究模型构建以及白血病复发防治等方面取得有潜在应用价值和创新性成果。