双重多肽介导三氧化二砷纳米粒靶向骨髓干细胞龛治疗慢性粒细胞白血病的研究

Chronic myeloid leukemia (CML) is one type of clonogenic hematopoietic stem cell diseases originated from bone marrow. Although BCR-ABL tyrosine kinase inhibitors are remarkably effective in inducing remission in chronic phase patients, they are not curative in a majority of patients due to their failure to eradicate leukemia stem cells (LSCs) inside bone marrow stem cell niches. Therefore, how to deliver therapeutic agent into stem cell niches to eradicate LSCs is the key to cure CML. Arsenic trioxide can remove LSCs via its multiple pharmacological activities such as autophagic degradation of BCR-ABL protein, priming LSCs differentiation and destroying the sanctuary of stem cell niches etc. Here, we construct a novel dual-peptide modified nanocarrier to target bone marrow stem niches according to the Chinese medicine theory of treatment aiming at its pathogenesis. We encapsulate As-Ni transitional metal compound into dual-peptide conjugated nanoparticles based on reverse micelles rationale. The loading density and stability of arsenic trioxide in nanoparticles are improved. The dual-peptide conjugated nanoparticles have the ability to deliver arsenic trioxide into stem cell niches including endosteal niches and vascular niches. Trivalent arsenic can be released via As-Ni compound dissociation among acidic conditions of endosomes after cellular uptake of nanoparticles inside niches. The CML-LSCs are finally eradicated by trivalent arsenic via its combined molecular activities. This stem cell niches targeted delivery strategy has the potential to be used in other malignant hematologic disorders treatment.

慢性粒细胞白血病（CML）是一种造血干细胞恶性克隆性疾病，病位在骨髓。骨髓干细胞龛中白血病干细胞的存在导致BCR-ABL抑制剂虽能有效缓解CML慢性期患者症状但不能治愈CML，如何有效清除干细胞龛中的白血病干细胞成为治愈CML的关键科学问题。砒霜主要成分三氧化二砷能通过降解BCR-ABL蛋白、促进白血病干细胞分化和克服干细胞龛庇护等多种药理机制清除白血病干细胞。本项目基于中医治病求本原则构建一种新型靶向骨髓干细胞龛的双重多肽修饰纳米给药系统。采用反胶束原理将三氧化二砷以砷镍过渡态结晶形式包载于聚合物纳米粒中，提高载药效率和稳定性；借助双重多肽介导将载药纳米粒递送到两类干细胞龛：骨内膜龛和血管龛；龛内纳米粒被细胞摄取后砷镍结晶在内涵体酸性环境释放三价砷，通过其综合药理机制有效清除白血病干细胞，解决当前CML治疗关键难题，为利用干细胞龛靶向策略治疗其它骨髓恶性肿瘤提供新思路。

由于骨髓干细胞龛中存在少量控制慢性粒细胞白血病（CML）发生的白血病干细胞，尽管BCR-ABL抑制剂能有效缓解CML慢性期患者症状但不能清除骨髓干细胞龛内的白血病干细胞治愈CML。中药砒霜主要成分三氧化二砷可通过克服干细胞龛对慢性粒细胞白血病干细胞庇护、降解BCR-ABL蛋白、促进白血病干细胞分化等综合机制发挥药效。本项目利用反胶束原理采用复乳法（w/o/w）成功将三氧化二砷以砷镍过渡态金属复合物结晶形式包载于PEG5k-PLGA20k聚合物纳米粒（～210 nm）中，纳米粒中包载的砷镍结晶尺寸在10～20 nm，和仅包载三价砷的纳米粒相比显著提高其三价砷包载量；通过对聚合物纳米粒表面DSS6和F56两种多肽比例优化，最终制备的干细胞龛靶向纳米粒表面DSS6多肽修饰量为50%，F56多肽为20%，此干细胞龛靶向纳米粒具有良好的骨内膜龛和血管龛靶向能力。干细胞龛靶向纳米粒在小鼠体内能够浓集在骨内膜龛所在的股骨和胫骨的松质骨内以及血管龛所在的骨髓血管内皮细胞附近，证明了其体内干细胞龛（骨内膜龛和血管龛）的靶向性；通过体外载药纳米粒与低结晶度羟基磷灰石（HA）组成的干骺端骨共孵育以及由CML细胞和血管内皮细胞形成的3D细胞球模型，评价了包载砷镍结晶的干细胞龛靶向纳米粒对白血病细胞分化和增殖的影响，50%的DSS6多肽修饰纳米粒显著降低了白血病细胞集落的大小和数量；20%的F56多肽修饰能促进HUVEC细胞对载药纳米粒的摄取，且可通过HUVEC细胞抑制白血病细胞的增殖；体内安全性评价结果表明，载药干细胞龛靶向纳米粒能显著降低由于三价砷在组织以及骨髓的非特异性分布造成的肝肾和骨髓毒性。本研究为利用干细胞龛靶向策略治疗其它骨髓恶性肿瘤提供了新思路。