CN114588111A - 一种酸敏性释药的三氧化二砷靶向脂质体组合及其制备方法 - Google Patents
一种酸敏性释药的三氧化二砷靶向脂质体组合及其制备方法 Download PDFInfo
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- A61P35/02—Antineoplastic agents specific for leukemia
Abstract
本发明属于医药技术和纳米医学技术领域,涉及一种酸敏性释药的三氧化二砷靶向脂质体组合及其制备方法和应用。本发明的装载三氧化二砷的脂质体载体由磷脂和胆固醇制备成,通过主动载药法装载三氧化二砷,具有长循环和酸敏性释药的特点;通过转铁蛋白修饰脂质体表面,进一步具有白血病细胞特异性摄取和胞内溶酶体内定位释药的特点,能有效发挥促急性早幼粒细胞白血病细胞凋亡和分化的作用,及抑制其他髓系白血病的功能。与现有技术的缓慢输注的普通亚砷酸注射液相比,本发明的三氧化二砷脂质体通过一次性静脉注射可达到增效减毒的疗效,并能显著改善患者的用药顺应性。
Description
技术领域
本发明属医药技术和纳米医学技术领域,涉及一种靶向白血病细胞、酸敏感释药的三氧化二砷脂质体组合及其制备方法和应用。
背景技术
三氧化二砷(Arsenic trioxide,ATO)是治疗急性早幼粒细胞白血病(Acutepromyelocytic leukemia,APL)的一线治疗药物,同时具有抑制白血病细胞增殖以及促进早幼粒细胞分化的作用。研究显示,低浓度的ATO可以直接降解PML-RARα融合蛋白,逆转对PML和RARα正常功能的抑制,从而促进APL细胞的正常分化;较高浓度的ATO会诱导细胞内活性氧的形成并减少谷胱甘肽的含量,导致APL细胞的细胞DNA和RNA损伤。然而,游离的ATO静脉注射后会从血液中快速清除,若是增加ATO的剂量以满足治疗要求又会造成对全身重要器官的严重毒性,特别是对心脏和肝脏的毒性。因此,在临床给药中需要以较慢的速度静脉内滴注亚砷酸盐以控制恒定的血药浓度,使功效最大化并使毒性最小化。目前,对于成年患者,将10mg的ATO稀释于500mL的5%葡萄糖注射液中,每日分2次输注,每次以8滴/分钟的速度进行输注,约l8~20h输注完成,两次间隔2h,四周为一疗程,间歇1~2周,尽管该滴注方法可以满足ATO有效剂量的持续给药,但过长的滴注时间给患者造成了极大痛苦,导致病人顺从性极差,且血药浓度一过性过高也会对正常组织造成严重的损伤。
脂质体是由磷脂和胆固醇为膜材制备的具有脂质双分子层结构的纳米尺度或微米尺度的封闭囊泡。脂质体可同时包载亲水性药物和亲脂性药物,提高药物体内外稳定性;降低药物体内清除速度,延长药物作用时间;改善药物生物利用度;使药物具有定向分布的靶向性,进而增强药理作用,降低药物毒性等诸多优点,被广泛运用于药物递送系统中。
转铁蛋白(Transferrin,Tf)是一种由79个氨基酸残基组成的血清糖蛋白,也是一种具有血清铁转运功能的内源性螯合物。Tf在pH 7.4的生理条件下,能够高度亲和Fe3+离子,每个转铁蛋白能结合两个铁离子。CD71,也被称为转铁蛋白受体1(Transferrinreceptor-1,TfR-1),广泛表达于人体各组织和细胞中,且其表达水平与细胞增殖代谢情况呈正相关。含铁转铁蛋白和细胞膜上的TfR1结合。Tf-TfR1复合物经转铁蛋白内吞途径进入细胞。当复合物遇到胞内质子泵效应或被转运至溶酶体,酸性pH环境使得Tf构象改变,释放铁离子以供细胞生长增值需要,该途径是细胞摄入铁元素的主要方式。TfR1在恶性增殖细胞表面的表达水平升高,以此满足细胞生长增殖对铁的大量需求。TfR1相对特异性强,且不易脱落,被认为是一种有效的癌症标志物,故而Tf介导的肿瘤靶向治疗,能在提高治疗效果的同时减少了对正常细胞的伤害,应用前景广泛。在白血病的靶向治疗中,CD71也被认为是有效的靶点之一。据报道,Tf修饰的载有紫杉醇的脂质纳米粒在体外能够高效地靶向人急性早幼粒细胞白血病HL-60细胞。
鉴于ATO狭窄的治疗窗,普通ATO注射液存在循环系统中快速清除、安全性、有效性和患者顺应性差等缺点,基于现有技术的现状,本申请的发明人拟提供一种靶向白血病细胞、酸敏感释药的三氧化二砷脂质体组合及其制备方法和应用。本发明将能够有效避免目前亚砷酸注射液在临床应用中的严重缺陷,具有较显著的临床应用前景。
发明内容
本发明的目的在于基于现有技术的现状,提供一种靶向白血病细胞、酸敏感释药的三氧化二砷脂质体组合及其制备方法和应用。
具体地,本发明提供了一种转铁蛋白修饰和未修饰的装载三氧化二砷的脂质体组合(ATO-LP和Tf-ATO-LP)及其制备方法,本发明可解决ATO注射液输注时间过长、患者顺应性低,个体差异大等应用受限问题,可实现治疗白血病的高效性、安全性和顺应性。
本发明是通过如下技术方案来实现的。
本发明所涉及的ATO-LP和Tf-ATO-LP,其结构为:脂质体的膜材由磷脂和胆固醇组成,水溶性药物ATO经金属盐溶液梯度法载于脂质双分子层内水相中,制得ATO-LP,进一步将Tf修饰在膜材表面,制得Tf-ATO-LP(如图1所示)。
本发明中,所述的ATO-LP和Tf-ATO-LP,其特征在于,构成脂质体膜的磷脂为天然磷脂、半合成磷脂和全合成磷脂中的一种或多种。其中,所述的天然磷脂为大豆磷脂和蛋黄卵磷脂中的一种或多种;所述的半合成或全合成磷脂包括磷脂酰胆碱类(PC)、磷脂酰甘油类(PG)、磷脂酰乙醇胺类(PE)或PEG化磷脂类中的一种或多种。其中,较佳的有大豆卵磷脂(SPC/EPC)、氢化大豆卵磷脂(HSPC),二硬脂酰磷脂酰胆碱(DSPC)、二油酰基卵磷脂(DOPC)、二硬脂酰磷脂酰甘油(DSPG)、二硬脂酰磷脂酰乙醇胺(DSPE)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-MPEG)中的一种或多种。
本发明中,所述的ATO-LP和Tf-ATO-LP中,磷脂与胆固醇的质量比为1:0.1~1:1,较佳的为1:0.2~1:0.5。
本发明中,所述的ATO-LP和Tf-ATO-LP中,磷脂与ATO的质量比为1:0.01~1:0.1,较佳的为1:0.02~1:0.05。
本发明中,所述的金属盐溶液为醋酸镍水溶液,浓度为200~600mM,较佳浓度为250~450mM;
本发明中,所述的Tf-ATO-LP中,Tf-PEG化的磷脂酰乙醇胺(PE)类磷脂复合物与脂质体的质量比为1:200~1:800,较佳的为1:400~1:600。
按上述方法制备ATO-LP或Tf-ATO-LP,体外稳定性与释放实验测定结果表明,Tf-ATO-LP与ATO-LP的粒径和电位无显著变化,并且两者具有相同的酸敏性释药特性,在血清中能保持稳定。体外细胞实验结果表明,白血病细胞摄取ATO-LP和Tf-ATO-LP的量显著高于正常白细胞和红细胞,与ATO-LP相比,Tf-ATO-LP被白血病细胞摄取量更高,抗白血病细胞的活性更大。在皮下异位瘤模型和原位模型药效试验中表明,与游离ATO相比,ATO-LP和Tf-ATO-LP在两种动物模型中均具有更好的抑制白血病作用和安全性,并且Tf-ATO-LP的疗效最好。
本发明提供的一种Tf修饰和未修饰的包载ATO的脂质体组合具有以下显著优点:
(1)以PEG化膜材制备脂质体,使脂质体具有体内长循环的作用效果,大幅延长ATO在体内的滞留时间。
(2)通过醋酸镍主动载药法,将ATO包载于脂质体的内水相中所制备的ATO-LP,呈现显著的酸触发释药的特征。
(3)该ATO脂质体能被白血病HL-60细胞非特异性摄取,而正常白细胞和红细胞对该脂质体的摄取量极有限,表现出白血病细胞摄取显著高于正常血细胞和白细胞的特征。
(4)以具有明确白血病细胞亲和性的Tf修饰在脂质体膜材外侧,可制成具有CD71靶向性的脂质体载体,(如图1所示)。同样方法包载ATO后,Tf-ATO-LP能够有效靶向包括急性早幼粒细胞白血病细胞在内的多种白血病细胞,进一步提高未修饰ATO脂质体被白血病细胞摄取的效率,进而可大幅提升ATO用药的疗效和安全性。
本发明提供了一种兼具长循环、被动或主动靶向白血病细胞和酸敏性释药功能的三氧化二砷脂质体,即ATO-LP和Tf-ATP-LP。该ATO脂质体经一次性静脉注射,一方面通过体内长循环和正常生理pH环境下几乎不释药的特性,可克服普通砷注射液长时间慢速输注所导致的患者顺应性差和个体差异大的缺点;另一方面,通过白血病细胞非特异性摄取ATO-LP或特异性摄取Tf-ATO-LP的机制,进入胞内溶酶体的脂质体在酸性环境下触发ATO释放,使得ATO发挥可靠且安全的疗效。因此,本发明所提供的ATO脂质体,有效避免了目前亚砷酸注射液在临床应用中的严重缺陷,具有极高的临床应用前景。
附图说明
图1为装载ATO的Tf-ATO-LP的结构示意图。
图2为ATO-LP和Tf-ATO-LP在不同缓冲溶液及血清中的稳定性考察,72小时内,ATO-LP和Tf-ATO-LP在不同缓冲溶液及血清中的粒径、PDI和ζ电位无明显变化,表明其稳定性良好。
图3为ATO-LP和Tf-ATO-LP的体外释放曲线,表明ATO-LP和Tf-ATO-LP具有相似的释放行为,呈现酸敏性释放特征,药物在正常组织中泄露率低。
图4为荧光素标记的Tf-LP与LP的细胞摄取程度,考察HL-60细胞的细胞摄取,表明HL-60细胞对荧光素标记的Tf-LP的摄取最高。
图5为ATO-LP和Tf-ATO-LP对HL-60细胞的体外毒性考察,横坐标所示药物浓度为ATO的浓度,结果表明Tf-ATO-LP相比ATO-LP具有更强的活性。
图6为ATO-LP和Tf-ATO-LP对HL-60细胞凋亡通路相关因子Caspase-3的影响,结果显示两种脂质体组的Caspase-3酶活力是游离药物组的3倍,而且Tf-ATO-LP组最高,表明ATO脂质体具有促细胞凋亡的活性,且显著高于游离药物。
图7为香豆素-6(Cou6)标记的Tf修饰和未修饰的脂质体Cou6-LP和Tf-Cou6-LP加入HL-60细胞和外周血白细胞(Peripheral white blood cell,PWBC)混合体系的细胞摄取程度,结果表明,在混合体系中,两种脂质体被HL-60的摄取量是PWBC的5倍,表明两种脂质体均对白血病细胞具有明显的选择性。
图8为ATO-LP和Tf-ATO-LP对HL-60细胞的诱导分化能力考察,横坐标所示药物浓度为ATO的浓度,结果表明Tf-ATO-LP相比ATO-LP具有更强的诱导分化作用。
图9为ATO-LP、Tf-ATO-LP与游离ATO的药动学曲线,结果表明本发明的ATO-LP和Tf-ATO-LP药动学行为相似,均具有体内长循环的特性。
图10为ATO-LP、Tf-ATO-LP与游离ATO的体内皮下异位瘤抗白血病考察,结果表明Tf-ATO-LP具有最强的抑制皮下瘤作用,能够显著抑制皮下瘤的生长。
具体实施方式
下面结合具体实例对本发明加以进一步的说明,但是不限制本发明的内容。下列实施实例中未注明具体条件的实验方法,按常规方法和条件操作。
实施例1薄膜水化法制备ATO-LP
称取16mg DSPC,4mg胆固醇,5mg DSPE-PEG2000,溶于氯仿/甲醇(4/1,v/v)中;50℃水浴中旋转蒸发去除有机溶剂,使其在烧瓶底部形成一层均匀的膜,加入醋酸镍水溶液,60℃旋转水化1小时,冰水浴中120W超声20分钟,使用柱层析法将脂质体外水相置换成氯化钠溶液,得到纳米脂质体悬液,加入三氧化二砷溶液,60℃水浴搅拌30分钟,使用G-50葡聚糖凝胶柱去除游离药物,得到ATO-LP。制得的脂质体粒径为110~120nm,电位为-8mV~-12mV,ATO含药量大于2%(wt/wt)。
实施例2转铁蛋白-PEG化的磷脂酰乙醇胺(PE)类磷脂复合物的制备
称取5.78mg DSPE-PEG2000-COOH溶于10mL MES缓冲液(含2mM EDC·HCl和5mMSulfo-NHS)中,在室温下搅拌30分钟;调节pH至7.2-7.5,加入10mg Tf,在室温下继续搅拌2小时;将反应液封装在截留分子量为14kDa的透析袋中,放入水中透析过夜,透析后冻干,得到DSPE-PEG2000-Tf复合物。
实施例3薄膜水化法结合后插入法制备Tf-ATO-LP
称取16mg DSPC,4mg胆固醇,5mg DSPE-PEG2000,溶于氯仿/甲醇(4/1,v/v)中;50℃水浴中旋转蒸发去除有机溶剂,使其在烧瓶底部形成一层均匀的膜,加入醋酸镍水溶液,60℃旋转水化1小时,冰水浴中120W超声20分钟,得到未经修饰的脂质体。再取DSPE-PEG2000-Tf复合物溶于脂质体悬液中,60℃水浴搅拌30分钟,使用柱层析法将脂质体外水相置换成氯化钠溶液,得到Tf-LP脂质体悬液。最后,加入三氧化二砷溶液,60℃水浴搅拌30分钟,使用G-50葡聚糖凝胶柱去除游离药物,得到Tf-ATO-LP。制得的脂质体粒径为110~120nm,电位为-8mV~-12mV,ATO含药量为2.03%(wt/wt)。
实施例4脂质体在不同缓冲液和血清中的稳定性考察
分别取ATO-LP和Tf-ATO-LP分别置于不同pH的PBS缓冲液或10%胎牛血清中,37℃恒温震荡孵育。于固定时间点测量粒径,考察其稳定性。结果如附图2所示,72小时内,ATO-LP和Tf-ATO-LP在PBS缓冲液或10%胎牛血清中的粒径均无明显变化,表明稳定性良好,且血浆蛋白结合可忽略不计。
实施例5脂质体的体外释放测定
分别取ATO-LP和Tf-ATO-LP封装在透析袋中,以不同pH的PBS缓冲溶液作为释放介质,37℃恒温振荡。于固定时间点,取透析袋外液,使用原子荧光测定法测定砷元素含量,计算累计释放百分率。结果如附图3所示,孵育72h后,在pH 4.5的PBS中,ATO-LP和Tf-ATO-LP的累积释放百分率均超过70%,分别是在pH 5.0(18.19%和19.51%)和pH 7.4(10.54%和10.42%)介质中的3倍和6倍。表明ATO脂质体在正常生理pH下呈现极低的释放量,而在酸性环境中能较快速地释出,表现出较好的酸敏性触发释药的特征。
实施例6脂质体的体外细胞摄取考察
取处于对数生长期的HL-60细胞,以1×106个/孔接种于24孔板。分别加入含0.3μg/mL香豆素6的Tf-LP、LP与游离香豆素6,继续孵育2小时后用4℃、pH 7.4的PBS清洗三次,使用流式细胞仪测量细胞的平均荧光强度。考察结果如图4所示,Tf-LP组中细胞的荧光强度最强,且预孵育Tf的细胞对Tf修饰的脂质体的摄取明显减小,表明Tf修饰能够显著提高细胞对脂质体的摄取。
实施例7体外毒性考察
取对数期生长的HL-60细胞铺于96孔板中,每孔1×104个,加入不同浓度的含药培养基,继续培养72h,加入10μL CCK-8试剂,孵育4h后,使用酶标仪分别于450nm及630nm下读取吸光度值,计算IC50值。考察结果如附图5所示,Tf修饰脂质体的IC50是未修饰的脂质体的2.7倍,表明Tf的修饰能够显著提高脂质体对白血病细胞的毒性。
实施例8体外促凋亡活性考察
取对数生长期的HL-60细胞铺于24孔板中,每孔1×106个,分别加入含不同浓度的游离ATO、空白脂质体或载药脂质体(ATO-LP、Tf-ATO-LP)的培养基,使药物终浓度达25μM,孵育12h。经3000rpm离心2min后,用PBS重悬,再离心,弃去上清液,收集细胞,加入50μL裂解液重悬沉淀下来的细胞,置于冰浴中裂解15min。于4℃,离心,将上清液转移至预冷的离心管中,使用Caspase-3活性检测试剂盒检测细胞内总Caspase-3水平。结果如附图6所示,ATO脂质体具有促细胞凋亡的活性显著高于游离药物。
实施例9正常外周血白细胞(PWBC)和白血病细胞对脂质体的摄取差异
取HL-60细胞加入到裸鼠外周血中,使HL-60细胞数达到10×109个/L,即高危APL病人外周血早幼粒细胞的最低浓度,随后与包载Cou-6的脂质体孵育,使用PE-anti humanCD45标记人源HL-60细胞,裂解红细胞,收集鼠外周血白细胞或者HL-60细胞后,使用流式细胞仪检测。结果如附图7所示,两种脂质体均对白血病细胞具有明显的选择性。
实施例10体外诱导细胞分化能力考察
取对数生长期的HL-60细胞铺于24孔板中,每孔1×106个,分别加入不同浓度的含药培养基,使药物终浓度达0、0.5、1、5、10、25μM,孵育5天后经3000rpm离心2min后,用PBS重悬洗涤细胞三次。加入APC anti-human CD11b,于室温下避光孵育30min,用PBS洗涤细胞,使用流式细胞仪检测CD11b+细胞比例。结果如附图8所示,脂质体处理后的HL-60细胞,分化程度与浓度正相关,且Tf修饰的脂质体处理的HL-60细胞中,CD11b+细胞比例显著高于ATO-LP。表明,Tf的修饰能够提高脂质体对HL-60细胞的诱导分化能力。
实施例11脂质体大鼠体内药动学考察
将9只雄性SD大鼠随机分为FreeATO、ATO-LP、Tf-ATO-LP三组,每组3只。按照1mg/kg体重的剂量尾静脉注射药物。给药后分别于5min、15min、30min、1h、2h、4h、8h、12h、24h、48h、72h后分别经眼眶取血0.5mL,置于肝素钠预饱和的离心管中。处理全血样品后,测定ATO含量。结果如附图9所示,与游离药物相比,ATO-LP与Tf-ATO-LP在体内的半衰期分别提高了约64倍与88倍,AUC0-t均增大了7倍,两者的MRT0-t是游离药物的7倍。表明ATO-LP和Tf-ATO-LP均具有缓释作用,能延长药物在体内的循环时间,在一定时间内能将血药浓度维持在一个稳定的水平。
实施例12体内药效学考察
取Nude雄性裸鼠随机分组,每组6只,建立HL-60皮下瘤模型,当肿瘤长至100mm3时开始给药。分别经尾静脉注射生理盐水、ATO、ATO-LP和Tf-ATO-LP,药物剂量为0.5mg/kg体重,每隔三天注射一次,记录肿瘤体积的变化。结果如附图10所示,在各给药组中,Tf-ATO-LP显示出最强的肿瘤抑制效果,Tf的修饰使脂质体的抑瘤率提高1.42倍。
Claims (11)
1.一种装载三氧化二砷的靶向脂质体组合,其特征在于,所述的脂质体的膜材由磷脂和胆固醇组成,脂质体表面修饰转铁蛋白,装载抗白血病药物三氧化二砷,其组成通式为:
ATO-LP和Tf-ATO-LP
其中,ATO为三氧化二砷(Arsenic trioxide),Tf为转铁蛋白(Transferrin);LP为脂质体载体(Liposome)。
2.按权利要求1所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的磷脂包括天然磷脂、半合成磷脂和合成磷脂,选自大豆卵磷脂、蛋黄卵磷脂、氢化大豆卵磷脂(HSPC)、磷脂酰胆碱(PC)类、磷脂酰甘油(PG)类、磷脂酰乙醇胺(PE)类或PEG化磷脂类中的一种或多种。
3.按权利要求1所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的ATO-LP通过如下方法制备:
将磷脂、胆固醇溶于有机溶剂中,得到的脂质溶液在40℃~80℃下减压旋转蒸发,除去有机溶剂后成膜,加入金属盐溶液作为水化介质,于20℃~80℃水浴中旋转或震摇水化,得粗制的脂质体悬液,经高压均质、超声或挤压法制得纳米尺度的小单室脂质体;最后将三氧化二砷溶液与脂质体悬液混合,在40℃~80℃下搅拌20~60分钟,采用超滤法、离心法、透析法或体积排阻色谱法去除游离药物,制得ATO-LP。
4.按权利要求1所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的Tf-ATO-LP通过如下方法制备:将磷脂、胆固醇溶于有机溶剂中,得到的脂质溶液在40℃~80℃下减压旋转蒸发,除去有机溶剂后成膜,加入金属盐溶液作为水化介质,于20℃~80℃水浴中旋转或震摇水化,得粗制的脂质体悬液,经高压均质、超声或挤压法制得小单室脂质体;再将转铁蛋白-PEG化的磷脂酰乙醇胺(PE)类磷脂复合物溶于小单室脂质体悬液中,在40℃~80℃下搅拌20~60分钟,采用超滤法、离心法、透析法或体积排阻色谱法,置换脂质体外水相,制得转铁蛋白修饰的纳米脂质体悬液;最后将三氧化二砷溶液与转铁蛋白修饰的纳米脂质体悬液混合,在40℃~80℃下搅拌20~60分钟,采用超滤法、离心法、透析法或体积排阻色谱法去除游离药物,制得Tf-ATO-LP。
5.按权利要求3或4所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的磷脂与胆固醇的质量比为1:0.1~1:1,较佳的为1:0.2~1:0.5。
6.按权利要求3和4所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的磷脂与三氧化二砷的质量比为1:0.01~1:0.1,较佳的为1:0.02~1:0.05。
7.按权利要求3和4所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的有机溶剂为醇类、烷烃、酮类、醚类、卤代烃中的一种或多种;所述的金属盐溶液为醋酸镍水溶液,浓度为200~600mM,较佳浓度为250~450mM;所述的超声操作为水浴超声或探头超声,较佳的超声功率为80W~200W,较佳的超声时间为5~20分钟。
8.按权利要求4所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的转铁蛋白-PEG化的磷脂酰乙醇胺(PE)类磷脂复合物按下方法述制备:将带有羧基的PEG化的磷脂酰乙醇胺(DSPE-PEG-COOH)溶于EDC·HCl/Sulfo-NHS反应液中,活化羧基,再将转铁蛋白溶于反应液中,与之发生缩合反应,制得转铁蛋白-PEG化的磷脂酰乙醇胺(PE)类磷脂复合物(Tf-PEG-DSPE)。
9.按权利要求4所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的转铁蛋白-PEG化的磷脂酰乙醇胺(PE)类磷脂复合物与脂质体的质量比为1:200~1:800,较佳的为1:400~1:600。
10.按权利要求3所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的ATO-LP体外具有缓释和酸敏感释药特征,体内具有长循环、白血病细胞非特异性摄取和胞内溶酶体定位释药特征。
11.按权利要求4所述的装载三氧化二砷的靶向脂质体组合,其特征在于,所述的Tf-ATO-LP,体外具有缓释和酸敏感释药特征,体内具有长循环、白血病细胞特异性摄取和胞内溶酶体定位释药特征。
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