CN114917357B - 转铁蛋白导向的装载plk1抑制剂的聚合物囊泡及其制备方法与应用 - Google Patents
转铁蛋白导向的装载plk1抑制剂的聚合物囊泡及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡及其制备方法与应用。本发明将PLK1抑制剂、聚合物混合、透析,得到装载PLK1抑制剂的聚合物囊泡,然后将装载PLK1抑制剂的聚合物囊泡与带有蛋白点击试剂的转铁蛋白反应,得到转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡,利用带负电的聚天冬氨酸内核有效装载Volasertib(TPVol)并能够体内靶向递送至MV‑4‑11 AML细胞并实现治疗。与自由药相比,TPVol具有更高的细胞毒性,PLK1抑制能力和体内的治疗效果,这主要是由于囊泡药物具有粒径有利于细胞摄取,循环稳定性高,生物相容性好,靶向性强等优点。以聚合物囊泡作为载体,开发靶向制剂,提高临床药物的治疗效果,是未来改善临床药物使用的有效尝试。
Description
技术领域
本发明属于药物技术,涉及聚合物载体药物,具体涉及转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡及其制备方法与应用。
背景技术
急性髓系白血病(Acute myeloid leukemia, AML)是异质性和克隆性造血干细胞(hematopoieiestem cells, HSC)疾病,通常表现为HSC异常克隆积累引起的遗传异常。尽管已经有越来越多的新技术譬如造血干细胞移植、激酶抑制剂、单克隆抗体或CAR-T细胞治疗等得到验证能够有效抑制AML,但是化疗仍然是治疗AML的主要方式之一。AML面临着诸如复发、耐药和严重副作用等严峻的挑战,并且约有40%的AML患者在达到缓解后最终复发并演变为难治性白血病,这也导致2020年全球所有癌症中AML死亡率高居第9位。最近,抗体、抗体-药物偶联物(ADC)和分子靶向药被越来越多地开发并用于AML治疗。ADC由于通常与高细胞毒性药物偶联即存在药物提前脱落的情况(譬如氧化石墨烯的脱落),具有明显的脱靶副作用和狭窄的治疗窗。而分子靶向药物如硼替佐米、来那度胺、Volasertib(BI6727)具有特异靶向性,在血液系统恶性肿瘤治疗中具有巨大的潜力。Volasertib是一种polo样激酶1(PLK1)抑制剂,已被美国食品和药物管理局(FDA)指定为治疗AML患者的孤儿药。PLK1在包括AML在内的大量癌细胞中都过表达,但在未转化细胞和正常细胞中表达非常低。然而,由于Volasertib的疏水性和小分子结构,导致其生物利用度低、毒性温和以及耐药性都是Volasertib在临床上面临的问题。此外,Volasertib在高剂量条件下具有剂量限制毒性,可能引起巨核细胞发育不全和严重的血小板减少,这些也限制了Volasertib的临床应用。
发明内容
本发明针对AML的载Vol的聚合物纳米药物表现出了显著延长小鼠生存期和延缓的肿瘤生长能力,并伴随着较小的副作用,这表明该纳米药物在AML治疗中有巨大潜力。
本发明中,转铁蛋白功能化的具有不对称膜结构的囊泡Tf-CPs能够利用带负电的聚天冬氨酸内壳有效装载Volasertib(TPVol)并能够在体内靶向递送至MV-4-11 AML细胞并实现治疗。与PVol和自由Volasertib相比,TPVol能够显著增强细胞摄取能力和对肿瘤细胞的杀伤能力。药代动力学实验和抗肿瘤实验表明TPVol能够显著延长药物在体内的循环时间,并且与PVol和Volasertib相比提现出了更优越的抗肿瘤效果。TPVol的抗肿瘤效果相比Volasertib有明显的增强作用,揭示了纳米药物的巨大优势,这也可能是AML靶向治疗的一个发展方向。
本发明采用如下技术方案:
一种转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡,包括聚合物囊泡;所述聚合物囊泡内装载PLK1抑制剂;所述聚合物囊泡外接有转铁蛋白;所述聚合物囊泡中,聚合物的侧链含有双硫键。
本发明中,PLK1抑制剂为MLN0905、RO3280、GSK461364、BI2536、Volasertib(BI6727)中的一种或几种。
本发明中,聚合物囊泡由聚合物组装得到,聚合物的侧链含有双硫键,具体的,聚合物为N3-PEG-P(A-DTC)与PEG-P(A-DTC)-PAsp,A为TMC、LA或者CL;优选为N3-PEG-P(TMC-DTC)与PEG-P(TMC-DTC)-PAsp,进一步优选的,N3-PEG-P(TMC-DTC)的摩尔量为N3-PEG-P(TMC-DTC)与PEG-P(TMC-DTC)-PAsp总摩尔量的2~15%,优选2~10 %。DTC、TMC、LA、CL都为单体,对应单体的化学结构式分别如下,开环聚合形成重复单元:
PAsp为聚天冬氨酸链段,PEG为聚乙二醇链段,N3为叠氮基团。
本发明中,组装聚合物囊泡的聚合物为两亲性聚合物,其中,亲水链段的分子量为3~15 kg/mol,疏水链段的分子量为10~30kg/mol,优选的,亲水链段的分子量为3.5~10kg/mol,疏水链段的分子量为13~20kg/mol;聚天冬氨酸链段的分子量为0.8~3kg/mol。优选的,P(A-DTC)为疏水链段,PDTC链段的分子量占疏水链段总分子量的10%~35%。
本发明中,转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡的制备方法为,将叠氮聚合物囊泡装载PLK1抑制剂,然后修饰转铁蛋白,得到转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡(TPVol)。通过后修饰的方法,利用Tf-DBCO和N3-PVol的点击化学反应实现转铁蛋白后修饰。
本发明中,将PLK1抑制剂、聚合物混合、透析,得到装载PLK1抑制剂的聚合物囊泡,然后将装载PLK1抑制剂的聚合物囊泡与带有蛋白点击试剂的转铁蛋白反应,得到转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡。
本发明公开了上述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡在制备抗肿瘤药物中的应用,具体为抗血液瘤,更具体为抗急性髓系白血病。转铁蛋白修饰的聚合物囊泡装载PLK1抑制剂Volasertib,能够靶向AML细胞,诱导PLK1失活并导致细胞死亡,从而阻止AML细胞浸润荷瘤NSG小鼠的肝脏、脾脏、血液和骨髓并造成损伤,优选的,所述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡中,转铁蛋白的密度为1~12%,优选为2~10%。
本发明制备了TfR靶向的装载有PLK1抑制剂Volasertib的囊泡TPVol,载药量达到5.1wt.%,粒径在48-51 nm之间,具有出色的稳定性和还原响应性。在细胞层面验证了TPVol能够靶向并高效杀伤TfR过表达的MV-4-11细胞。在动物层面上首先验证了TPVol能够在体内长循环并最终靶向富集到归巢至骨髓的MV-4-11细胞,随即在治疗AML小鼠的实验中证实通过尾静脉注射TPVol能够有效抑制AML细胞增殖,显著减少AML细胞在器官内的浸润,延长AML小鼠中位生存期,并保护骨结构不被破坏。与自由Volasertib相比,TPVol具有更高的细胞毒性,PLK1抑制能力和体内的治疗效果,这主要是由于囊泡药物具有粒径有利于细胞摄取,循环稳定性高,生物相容性好,靶向性强等优点。以聚合物囊泡作为载体,开发靶向制剂,提高临床药物的治疗效果,是未来改善临床药物使用的有效尝试。
附图说明
图1为(A)Tf和Tf-DBCO的质谱图;(B)TPVol和PVol的粒径分布;(C)TPVol在4℃下放置7天和在10% FBS存在下37℃放置18 h的粒径分布;(D)新鲜制备的TPVol和放置7天的TPVol对MV-4-11细胞的毒性,赋予时间为72 h;(E)TPVol在10 mM DTT中孵育18 h期间的粒径分布。(F)Triton,TPVol和自由Volasertib的溶血实验,孵育时间3 h;(G)MTT法测定TPVol,PVol和自由Volasertib对健康细胞L929的细胞毒性。
图2为(A)流式细胞仪测试了Cy5-PVol和Cy5-TPVol在TfR高表达的MV-4-11细胞中孵育1 h后被内吞的情况; MTT法测定(B)不同靶向密度Tf的TPVol和自由Volasertib在MV-4-11细胞中孵育72 h的细胞毒性、(C)TPVol、PVol和自由Tf封闭后TPVol在MV-4-11细胞中孵育72 h的细胞毒性(D)TPVol和PVol在697细胞中孵育72 h的细胞毒性;(E)流式细胞仪测定的药物浓度0.07 μg/mL下TPVol,PVol和Volasertib孵育72 h对MV-4-11的凋亡情况、(F)Western blot测定TPVol,PVol和Volasertib与MV-4-11孵育72 h后细胞内PLK1,AKT,p-AKT和GAPDH水平检测和(G)半定量分析。
图3为(A)自由Volasertib,PVol和TPVol在健康Balb/c小鼠体内的药代动力学曲线(6 mg Volasertib/kg,n=3)和(B)曲线下面积分析;NSG小鼠接种MV-4-11-Luc-GFP细胞后(B)第6天和第12天的体内生物发光成像和(C)第12天AML细胞在小鼠体内的富集情况成像图和(D)肝脏,脾脏和骨髓用流式细胞术检测浸润情况;接种后第12天尾静脉注射Cy5标记的TPVol和PVol,8 h后在小鼠(E)后腿股骨和胫骨的富集荧光成像、(F)心脏、肝脏、脾脏、肺、肾脏的富集荧光成像和(G)半定量分析,n=3。
图4为TPVol,PVol和自由Volasertib对MV-4-11-Luc-GFP原位小鼠模型的体内抗肿瘤实验(n=7)。(A)接种与给药方案;(B)给药后小鼠的生物发光图像与(C)定量分析;(D)小鼠的相对体重;(E)小鼠生存期曲线。
图5为流式细胞术测定的经过治疗后小鼠的肝脏、脾脏、骨髓和外周血中AML细胞含量的(A)流式散点图和(B)定量分析。
图6为PBS、自由Volasertib、PVol,4% TPVol和高剂量4% TPVol治疗荷AML小鼠14天后小鼠(A)主要脏器和腿骨切片的H&E染色图,标尺为50 μm;(B)骨髓中细胞PLK1和p-Akt表达水平和(C)半定量分析。
图7为PBS、自由Volasertib、PVol,4% TPVol和高剂量4% TPVol治疗荷AML小鼠14天后小鼠后腿股骨和胫骨的(A)TRAP染色切片;(B)μCT重建图像和(C)μCT定量分析。
图8为Ps-Vol,高剂量自由Volasertib,自由Volasertib和PBS对MV-4-11-Luc-GFP原位小鼠模型的体内抗肿瘤实验(n=6)。(A)给药后小鼠的生物发光图像与(B)定量分析;(C)小鼠的相对体重;(D)小鼠生存期曲线。
图9为转铁蛋白功能化的具有不对称膜结构的囊泡Tf-CPs能够利用带负电的聚天冬氨酸内核有效装载Volasertib(TPVol)并能够体内靶向递送至转铁蛋白受体(TfR)高表达的MV-4-11 AML细胞并实现治疗。
具体实施方式
原料、试剂及仪器
嵌段聚合物N3-PEG-P(TMC-DTC)(M n=7.5-(15.0-2.0) kg mol-1),PEG-P(TMC-DTC)-PAsp(M n=5-(14.1-2.2)-1.29 kg mol-1)和A6-PEG-P(TMC-DTC)(M n=0.9-7.5-(14.7-2.1)kg mol-1)为现有产品,也可以根据申请人之前公开的文献或者专利申请常规制备。Volasertib(Vol,≥ 99.8%, MedChemExpress, USA),转铁蛋白(Tf, ≥ 98 %, Sigma-Aldrich),A6为多肽(Ac-KPSSPPEEC-NH2,98%)购自上海吉尔生化,末端带有巯基,聚天冬氨酸(KD10,98%,上海强耀生物),NHS-OEG4-DBCO(97%, BrodPharm),谷胱甘肽(GSH,>99%,Roche),1,4-二硫代-D,L-苏糖醇(DTT,>99%,Merck),Cy5-NHS(Lumiprobe,USA),4′,6-双脒基-2-苯基吲哚(DAPI, 碧云天),D-荧光素钾盐(99%, Bridgen Co., Ltd.)和N-2-羟乙基哌嗪-N-2-乙磺酸(HEPES,ThermoFisherScientific)均购买后直接使用。二甲基亚砜(DMSO)购自国药集团,经无水硫酸镁干燥、减压蒸馏后除水使用。Polo样激酶(PLK1)抗体和GAPDH抗体(Santa Cruz Biotechnology),牛血清蛋白(BSA, fraction V, heat shockisolation,Bio Basic Inc),人CD45抗体(anti-CD45-APC, BD Biosciences),细胞计数试剂盒-8(CCK-8,Dojindo Laboratories),BCA试剂盒(Thermo Scientific),和Annexin V-F647/PId凋亡试剂盒(Fcmacs Biotech, Nanjing)均购买后按说明书使用。MV-4-11人源AML细胞从ATCC购买,MV-4-11-Luc-GFP细胞由苏州大学附属第一医院提供。
DBCO和Tf的接枝率由基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS,Bruker Daltonics Ultraflex II)测定。聚合物囊泡的粒径和粒径分布通过配有He/Ne激光光源(波长为633 nm)和173°背折射检测器的ZetaSizer Nano-ZS纳米粒度仪(MalvernInstruments)测定,温度为25℃。多功能酶标仪(VarioskanLUX,Thermo Scientific)用来进行蛋白浓度的BCA检测。酶标仪(Thermo Multiskan FC)用来测定细胞与CCK-8反应的生成物在450nm处的吸光度值。Volasertib的浓度由紫外-可见光分光光度计(HitachiUH5300)测定。流式细胞仪(FACS Calibur,BD sciences)用来研究细胞内吞、细胞凋亡、细胞分选和检测并用Flowjo软件(TreeStar)进行分析。骨组织结构由微计算机断层扫描技术(μCT, SkyScan 1176, Aartselaar, Belgium)检测(65 kV, 385 mA,and a 1 mm Alfilter)。骨结构的三维重建、骨小梁体积分数、骨表面积分数、骨小梁厚度、骨小梁分离度和骨小梁个数由NRecon软件分析。体内生物发光成像和体外荧光成像由近红外荧光成像系统(IVIS, Lumina II; Caliper, MA, USA)拍摄。
动物模型
所有动物实验及操作均获得苏州大学实验动物中心和苏州大学的动物护理和使用委员会的批准。为了建立MV-4-11-Luc-GFP AML移植瘤模型,6周龄雌性NSG小鼠(22-24g)在175 cGy剂量下辐照,6 h后尾静脉注射0.02mLMV-4-11-Luc-GFP细胞(1×105个)。病程通过腹腔注射D-荧光素钾盐(50 mg/kg)10分钟后用近红外荧光成像系统拍摄检测。
本发明所有数据均是以平均值呈现,组间差异性由Anova单因素方差分析评估,生存曲线使用Kaplan-Meier和log-rank比较检验分析,*p<0.05视为具有显著性差异,**p<0.01,***p<0.001和****p<0.0001视为高显著性差异。
为了增强PLK1抑制剂Volasertib在针对AML治疗上的潜力,本发明设计制备了能够高效装载带有正电荷Volasertib的具有不对称膜结构,内腔带有负电荷,并且能够靶向TfR的囊泡TPVol。TPVol囊泡能够在血液中长时间循环,并快速地进入AML细胞达到高效杀伤肿瘤细胞的目的,从而消除或减少AML对脏器和骨的浸润和破坏,达到抑制病程,延长生存期的目的。研究了TPVol的制备和理化性质的表征,并且在细胞和动物层面研究了TPVol对AML移植模型的治疗效果。
实施例一 Volasertib囊泡的制备和表征
TPVol通过后修饰的方法,利用Tf-DBCO和N3-PVol的点击化学反应制备。为了制备Tf-DBCO,首先将41 μL的NHS-OEG4-DBCO的DMSO溶液(10 mg/mL)加入到2 mL的Tf水溶液(20mg/mL)中(Tf/DBCO=1/1.2),在37℃,200 rpm的环境下反应12 h,随后将反应液超滤离心三次(MWCO 10000 Da),得到Tf-DBCO,其溶液用质谱表征接枝率,用BCA测试Tf最终浓度。
制备N3-PVol。在37℃下将100 μL 含有N3-PEG-P(TMC-DTC)、4 mg Volasertib和PEG-P(TMC-DTC)-PAsp的DMSO溶液(N3-PEG-P(TMC-DTC)的摩尔量为N3-PEG-P(TMC-DTC)与PEG-P(TMC-DTC)-PAsp总摩尔量的2.5%)在300 rpm的搅拌下加入0.9 mL HEPES溶液(5 mM,pH 6.8)并继续搅拌10分钟,随后在37℃, 200 rpm的摇床中孵育2 h,并在HEPES(5 mM,pH7.4)中透析6 h(MWCO 14000 Da),每小时更换介质,得到理论载药量为9.1wt.%的N3-PVol,N3基团密度为2.5%。按上方法,N3-PEG-P(TMC-DTC)的摩尔量为N3-PEG-P(TMC-DTC)与PEG-P(TMC-DTC)-PAsp总摩尔量的5%、7.5%、10%时,N3-PVol中N3基团密度为5%、7.5%、10%。
制备TPVol。按Tf-DBCO/N3摩尔比1.8∶1,将Tf-DBCO加入N3-PVol溶液中,搅拌1分钟,随后放置于37℃,100 rpm的摇床中反应12 h,反应液通过超滤(MWCO 1 million Da)除去多余的Tf-DBCO,得到TPVol。
制备PVol。在37℃下将100 μL 含有4 mg Volasertib和PEG-P(TMC-DTC)-PAsp的DMSO溶液在300 rpm的搅拌下加入0.9 mL HEPES溶液(5 mM,pH 6.8)并继续搅拌10分钟,随后在37℃,200 rpm的摇床中孵育2 h,并在HEPES(5 mM,pH 7.4)中透析6 h(MWCO 14000Da),每小时更换介质,得到理论载药量为9.1 wt.%的PVol。
在上述PEG-P(TMC-DTC)-PAsp和N3-PEG-P(TMC-DTC) 聚合物中掺入0.5mol%的Cy5标记的PEG-P(TMC-DTC),其余不变,得到Cy5-TPVol;在上述PEG-P(TMC-DTC)-PAsp中掺入0.5mol%的Cy5标记的PEG-P(TMC-DTC),其余不变,得到Cy5-PVol。
为了研究TPVol的稳定性,载药囊泡在4℃下被保存在含有10% FBS的溶液中并在不同时间点检测其粒径和粒径分布。为了研究囊泡的还原环境响应性,载药囊泡溶液中在氮气保护下加入谷胱甘肽(GSH)溶液(10 mM),在37℃、200 rpm的摇床中放置,在不同时间点检测其粒径和粒径分布。纳米粒的粒径、粒径分布(PDI)和Zeta电位使用DLS测定。Volasertib的浓度使用紫外分光光度计检测在322 nm处的吸光度。并根据Volasertib在1~50 μg/mL范围内的标准曲线计算浓度,并根据现有方法计算载药量和载药效率。
通过点击化学反应将叠氮官能化囊泡N3-PVol与DBCO官能化的Tf(Tf-DBCO)键合,制备了装载Volasertib(Vol)的靶向聚合物囊泡TPVol。首先,通过N3-PEG-P(TMC-DTC)(分子量7.5-(15.0-2.0) kg/mol)和PEG-P(TMC-DTC)-PAsp (分子量5.0-(15.0-2.0)-1.29kg/mol)在摩尔比2.5/97.5、5/95、7.5/92.5或10/90下掺入9.1 wt.% Vol并且在HEPES缓冲溶液(5 mM,pH 6.8)中的自组装得到N3-PVol。DLS和UV的测试结果表明,制备得到的表面N3基团可控的N3-PVol具有小而均一的尺寸和稳定的载药量(表1),随着N3基团密度的增加,从46.3 nm增大到48.2 nm,载药量则稳定在5.2-5.1wt.%之间,这说明N3基团的密度对于囊泡的尺寸、分布和载药量没有明显影响,PAsp作为囊泡的内壳层带有负电荷,与带正电的Vol之间产生强静电相互作用和氢键作用,可以相对高效地促进Vol的装载(超过53%)。
随后通过Tf上的氨基与DBCO-OEG4-NHS的反应制备得到Tf-DBCO,分析了Tf/DBCO摩尔比在1/1.2和1/2时的反应效率。从质谱的测量结果和定量分析可以看到,Tf/DBCO摩尔比在1/1.2时已经达到了几乎100%的接枝率(图1 A),可以进一步用于N3-PVol的表面修饰。在室温下通过点击化学反应,在不同的Tf-DBCO/N3的摩尔比下,可以将Tf-DBCO偶联至N3-PVol上,得到Tf密度可控的TPVol。DLS和BCA的结果可以看出,当Tf-DBCO的投料比为2.5%,5.0%,7.5%或10.0%时(N3-PVol中N3基团密度为10%),最终可以得到Tf表面密度为2%,4%,6%或8%的TPVol,反应效率大致保持在82%-88%,并且Tf修饰几乎不影响囊泡的尺寸、尺寸分布和载药量(表2,图1 B)。
TPVol具有良好的稳定性,以N3-PVol中N3基团密度为10%、Tf表面密度为4%为例:在4℃下放置7天,粒径基本不变(图1 C)。并且为了模拟体内循环时的环境,在37℃,10% FBS的条件下测试了TPVol的稳定性,DLS的结果表明(图1 C),靶向囊泡在18 h的放置下粒径分布几乎没有变化,为囊泡能够在血液中稳定循环提供了佐证。进一步在细胞层面简单的检测了放置7天后囊泡的细胞毒性,实验结果证明放置7天的囊泡药物与新鲜制备的相比细胞毒性几乎没有差异(图1 D),进一步证明了囊泡的稳定性。囊泡能够在多种条件下保持稳定得益于聚合物中DTC的双硫戊环能够形成自交联,提高了囊泡的稳定性,且药物与聚合物的相互作用也提供有利效应。为了验证稳定的囊泡到达AML细胞内能够快速的响应并释放药物,模拟了肿瘤细胞内的高还原环境,在介质中添加了10 mM的GSH。从DLS的结果(图1 E)可以发现TPVol在10 mM GSH的还原条件下4 h会发生明显的溶胀,直至18 h囊泡结构几乎完全被破坏,与先前的稳定截然相反,说明自交联的双硫结构在还原环境下能够快速解交联并释放药物。综上,本发明的囊泡TPVol能够兼具能够在血液中稳定循环并在肿瘤细胞内快速释放的特性,为后期的细胞动物实验提供了依据。
MV-4-11是人源急性髓系白血病细胞,发病快,迅速致死,以MV-4-11细胞为研究对象,研究Tf靶向分子修饰的囊泡TPVol的安全性,靶向能力和细胞毒性的改变;如无说明,以N3-PVol中N3基团密度为10%、Tf表面密度为4 %为例。
实施例二
细胞毒性试验(CCK-8)
细胞选用人源急性髓系白血病细胞株MV-4-11、人源B淋巴细胞白血病细胞株697、健康小鼠成纤维细胞株L929和小鼠高转移性黑色素瘤细胞株B16F10。MV-4-11、L929和697用RPMI 1640培养基(Gibco)在5%的CO2培养,培养基中添加10%血清、1% L-谷氨酰胺、青霉素(100 IU/mL)和链霉素(100 g/mL)。B16F10细胞用DMEM培养基(Gibco)在相同条件下培养。
将MV-4-11细胞铺于96孔板(5 × 103个/孔,80 μL),24小时后细胞汇合率达到60%左右,分别加入20 mL PBS,TPVol,PVol和Volasertib孵育72 h,以Volasertib计,浓度范围为0.01到20 μg/mL。孵育72 h后,每孔加入10 mL CCK-8溶液孵育4 h,随后用酶标仪测定各孔在450 nm的紫外吸收。加入PBS的细胞作为对照组,细胞的存活率为各孔吸光度与对照组吸光度的比值。每个实验平行5次(n=5),最终呈现的结果为平均值±SD。对MV-4-11细胞的半数抑制浓度(IC50)通过非线性回归计算得到。
在验证纳米药物(TPVol,PVol)和Volasertib(自由药物)对697、L929和B16F10细胞的毒性实验中,选用各细胞对应的培养基,铺板的细胞数分别为5×103个、8×103个和3×103个每孔,孵育时间分别为72 h、4+68 h和4+68 h(+前为与药物孵育时间,+后为更换新鲜培养基后孵育时间),其余步骤与上述相同。
流式细胞仪试验
为了测定MV-4-11对囊泡的摄取情况,在上述PEG-P(TMC-DTC)-PAsp和N3-PEG-P(TMC-DTC) 聚合物中掺入0.5mol%的Cy5标记的PEG-P(TMC-DTC)聚合物并制备囊泡用于实验。将MV-4-11细胞铺于6孔板中(5×105个/孔),24小时后分别加入PBS,Cy5-PVol和带有不同Tf密度的Cy5-TPVol(1.0 μg Cy5/mL)。孵育1小时后,收集细胞在800 rpm下离心5 min并用PBS清洗2次,分散至500 μL PBS中用于FACS测试。
为了测定药物处理后MV-4-11的凋亡情况,MV-4-11细胞铺于6孔板中(3×105个/孔),24小时后分别加入PBS,Volasertib,PVol和TPVol(0.07 μg/mL Volasertib)。孵育48小时后收集细胞,800 rpm离心5 min并用PBS清洗2次,最终分散至100 μL binding buffer中(1×106个/mL),用凋亡试剂盒中提供的Annexin V-Alexa Fluor 647/碘化丙啶(PI)避光染色15min后,用400 μL binding buffer终止并用于FACS测试。
蛋白免疫印记(Western blot)实验
为了探究Volasertib制剂对细胞蛋白表达的影响,MV-4-11细胞铺于6孔板中(3×105个/孔),24小时后分别加入PBS,Volasertib,PVol和TPVol(0.07 μg/mL Volasertib)并孵育48 h。收集细胞,800 rpm离心5 min并用PBS清洗1次,用预冷的RIPA缓冲液裂解15min,并在4℃下1200 rpm离心取上清。将总蛋白浓度相同的上清液与loading buffer混合,煮沸5分钟,用10%SDS-PAGE分离(20 μg/孔),转移至聚偏二氟乙烯膜上进行电泳。完成后用5%脱脂牛奶在25℃下封闭1.5 h,条带分别用PLK1抗体、AKT抗体、p-AKT抗体和GAPDH抗体在4℃下孵育过夜。条带用含有0.1% 吐温-20的Tris缓冲液(TBST)洗涤3次,并与辣根过氧化氢酶标记的二抗在室温下孵育1.5 h。印迹图像通过条带在显影液处理后由电化学发光检测系统(Pierce)拍摄。
溶血实验
为了检测Volasertib和其制剂的溶血情况,取小鼠全血2 mL,用8 mL无菌PBS洗涤3次,每次洗涤后150 g离心5 min,弃上清,最终取得的红细胞悬液在无菌PBS中以体积比1:9稀释。细胞悬液与曲拉通-X(阳性对照),Volasertib和TPVol孵育3 h,150 g离心5 min,取上清液用紫外-可见光分光光度计测定在545 nm处的吸光度。
生物相容性是纳米药物最为重要的特性之一,通过溶血实验验证了TPVol的安全性。实验表明,自由的Volasertib在0.5 mg/mL时即引起明显的溶血,而4% TPVol在1mg/mL时对血细胞造成的溶血影响也较低(图1 F)。与此同时,在小鼠成纤维细胞L929上的细胞毒性实验也表明,PVol和TPVol在实验浓度下均对L929没有明显毒性(图1 G),而自由的Volasertib在10 μg/mL到40 μg/mL的浓度范围下对L929产生了明显毒性。
研究发现在高增殖性的AML细胞中,很多表面靶点都过表达,为靶向治疗AML提供了可能性,但是并非有受体表达即可实现高靶向。为了研究MV-4-11对囊泡的摄取,制备了Cy5标记的带有不同Tf表面密度(2%,4%,6%和8%)的囊泡Cy5-TPVol。流式细胞术表明,MV-4-11对所有TPVol的摄取都比对非靶向组的PVol更高,其中Tf表面密度为4%和6%的TPVol达到1.6倍的增强摄取的效果(图2 A),证明Tf对促进PVol被MV-4-11细胞内吞有明显的增强作用。
为了研究TPVol的细胞毒性,进行了对MV-4-11的细胞毒性实验。结果表明,TPVol在孵育72 h的条件下能够对MV-4-11细胞产生极高的细胞毒性,其中4% TPVol的IC50为0.07μg/mL,比PVol低2倍(图2 B)。这种增强效果高于TfR靶向的脂质体,并且使用自由Tf对MV-4-11细胞进行封闭后,Tf增强细胞毒性的效果消失,TPVol与PVol显示的细胞毒性接近,进一步证实了TPVol能够增强细胞毒性(图2 C)。在697细胞中,4% TPVol与PVol的细胞毒性没有差异(图2 D)。这些结果验证了TPVol在MV-4-11细胞中的主动靶向能力,在表面密度为4%-6%时,TPVol的细胞摄取和细胞毒性达到最大,但随着密度的进一步增加,被摄取能力和细胞毒性都没有明显增加。
进一步采用流式细胞术Alexa Fluor 647/碘化丙啶(PI)染色技术研究TPVol、PVol和Volasertib诱导MV-4-11细胞凋亡的能力。结果显示,自由Volasertib在0.07 μg/mL的浓度下细胞毒性较弱,仅导致14.9%的细胞凋亡,而TPVol和PVol的凋亡诱导明显增强,分别为77.8%和54.7%(图2 E),进一步证明了囊泡药物强细胞毒性的能力。
Polo样激酶1(PLK1)是一种丝氨酸/苏氨酸蛋白激酶,主要参与细胞的DNA复制、细胞应激反应和有丝分裂进程。研究发现,PLK1在多种癌细胞中过表达,包括乳腺癌、前列腺癌、肾癌和急性髓系白血病。而与此同时,PLK1在正常细胞或未转化细胞中的表达与癌细胞相比显著降低,这使得PLK1成为一个很好的靶点。研究表明,下调PLK1能够导致癌细胞周期阻滞并进一步导致凋亡,因此PLK1抑制剂作为优秀的抗癌药物已经得到了广泛的关注。为了评价TPVol对PLK1的体外抑制作用,对TPVol,PVol和Volasertib处理过的MV-4-11细胞进行了Western blot测试。实验表明,所有样品几乎没有抑制PLK1和AKT的表达,却显著抑制了磷酸化AKT(p-AKT)的表达。其中,TPVol表现出了明显的最强的抑制能力(图2 F&G)。据报道,Volasertib是一种ATP竞争性的PLK1抑制剂,通过与PLK1的三磷酸腺苷结合域特异性结合,导致PLK1的失活,并进一步导致细胞凋亡。Western blot的结果可能是由于PLK1诱导的PTEN蛋白磷酸化过程被PLK1抑制剂显著抑制,而PTEN作为致癌信号PI3K-AKT信号通路的负调控因子,可以阻断AKT的激活。PI3K-AKT通路对细胞的增殖至关重要,并且与有丝分裂细胞中PLK1的激活相关。结合以上实验结果,可以证明MV-4-11对TPVol内吞,导致增强的PLK1抑制,并导致增强的细胞毒性和凋亡。
实施例三
药代动力学实验
为了分析囊泡的药代动力学,使用FITC标记的Volasertib极其制剂进行研究,为常规技术。通过尾静脉将Volasertib-FITC,PVol-FITC和TPVol-FITC(6 mg Volasertib/kg)注入健康Balb/c小鼠体内(n=3),在预定时间点,用肝素化毛细采血管从小鼠内眦静脉丛取血70 μL,5000 rpm离心20 min,取20 μL血清加入0.1 mL 1% 的曲拉通并超声,再加入200 μL含20 mM DTT的DMSO,在37℃、200 rpm的摇床中过夜萃取Volasertib。随后离心后取上清,用荧光分光光度计测试。Volasertib浓度对时间作图得药代动力学曲线,并用Excel和Origin 8软件拟合得到半衰期(t1/2β)和曲线下面积(AUC)。
体内靶向能力和离体成像实验
为了评价TPVol的靶向能力和在骨髓中的富集程度,对小鼠使用了离体成像。在小鼠接种后第12天尾静脉注射Cy5标记的TPVol或PVol(6 mg Volasertib/kg)。注射后8 h处死小鼠,采集主要脏器,用IVIS成像系统进行离体成像,使用Living Image 2.6软件进行分析。
体外实验研究了TPVol对MV-4-11的细胞毒性及PLK1抑制情况,但囊泡药物能否长时间在血液中循环并到达AML细胞富集的部位,对于治疗AML来说至关重要。因此,研究了Volasertib及其囊泡制剂的药代动力学和体内分布情况。结果表明,TPVol与PVol在血液中有着相似的药代动力学行为,消除相半衰期(t1/2β)分别为10.7 h和12.9 h(图3 A),与自由的Volasertib的循环时间(t1/2β2.5 h)相比有着显著的延长,曲线下面积也显著增大(图3B,****p)。这说明聚合物囊泡对Volasertib能够有效地保护并延长循环时间,从而为体内的富集和较好的治疗效果提供可能性。
较长的循环时间为体内的富集提供了优秀的支撑,接着研究了TPVol在小鼠体内的富集情况。用NSG小鼠通过尾静脉注射MV-4-11-Luc-GFP细胞建立了原位AML模型,根据之前的AML建模研究,该模型中的AML细胞将主要聚集在小鼠的骨髓、肝脏和脾脏中(图3 C)。在接种后第12天,肝脏、脾脏和骨髓中的AML细胞浸润程度分别达到了12.4%,7.3%和57.8%(图3 D)。对建模后的第12天的小鼠分别尾静脉注射了Cy5标记的TPVol和PVol,并在8 h后处死小鼠。成像结果表明,与PVol组相比TPVol在后腿中富集的Cy5信号显著增强(**p,图3E),这说明接种AML细胞的小鼠的腿骨中归巢了大量AML细胞,导致TPVol能够靶向至腿骨中的AML细胞,从而在图像上显示出了明显增强的信号。然而TPVol和PVol纳米药物在主要器官的富集则没有明显差异(图3 F&G),成像结果表明在小鼠体内,TPVol也能够很好的发挥作用靶向至MV-4-11细胞,而前期的实验也证明即使囊泡富集于其他组织中,也不会对正常细胞产生明显的毒性。综合以上实验结果,TPVol表现出了对MV-4-11良好的靶向能力和细胞毒性,这为后续的动物实验提供了有力的证据。
实施例四 Volasertib囊泡的抗肿瘤活性
接种了MV-4-11-Luc-GFP的小鼠被随机分为7组,每组7只(n=7):PBS,Volasertib,PVol,2% TPVol,4% TPVol和6% TPVol,在第5,7,9和11天时尾静脉给药(6 mg Volasertib/kg),以及4% TPVol 在第5,8,11和14天尾静脉给药(9 mgVolasertib/kg)。在第5,8,11和14天腹腔注射D-荧光素钾盐(50 mg/kg),在注射的10-15分钟后用异氟烷进行吸入式麻醉并使用IVIS成像系统检测生物发光情况,用Living Image 2.6软件进行分析。每2天检测小鼠体重并观察生存期至第49天,绘制Kaplan-Meier生存曲线。
为了验证TPVol在体内治疗AML的能力,选用荷原位MV-4-11-Luc-GFP的NSG小鼠作为模型,研究了囊泡药物对AML病程的抑制能力。在第0天对小鼠进行1.75 cGy的辐照,并在辐照6小时后接种1×105个MV-4-11-Luc-GFP细胞完成建模。接种后第5天通过尾静脉分别注射带有不同靶向密度的TPVol、PVol、Volasertib(6mg Vol/kg)和PBS,每2天给药一次,共注射4次,以及4% TPVol进行更高剂量(9 mg Vol/kg,每3天给药一次,共注射4次)的治疗。在接种后第5、8、11和14天对小鼠进行生物发光成像,并对小鼠体重和生存期进行了持续的检测(图4 A)。
生物发光成像及定量分析显示(图4 B&C),MV-4-11-Luc-GFP随时间很快扩散至全身,PBS组从第5天到第14天的生物发光快速增强,肿瘤进展迅速,并且在腿部有明显的富集。MV-4-11-Luc-GFP AML模型极具侵袭性,小鼠体重随病程发展开始出现明显下降(图4D),并且第13天就开始出现小鼠死亡,到第15天时7只小鼠全部死亡,中位生存期仅14天(图4 E)。与之相比,自由Volasertib组展现出了对病程发展一定程度的延缓,在第14天时显著抑制了AML细胞的扩增(图4 C)。但由于自由Volasertib的剂量限制毒性,部分小鼠出现了体重下降,并且给药后小鼠尾巴长时间无法止血,可能是血小板减少的症状。但由于剂量相对较低,停止给药后逐渐恢复正常。在先期研究中,小鼠在15 mgVol/kg的剂量下两针后很快死亡(第5和第8天尾静脉注射),证明了自由Volasertib的毒性,最终的中位生存期延长至17天。PVol与自由Volasertib和PBS相比展现出了明显更优秀的抗肿瘤能力,并且所有纳米药物的给药对小鼠体重均没有明显影响,最终将中位生存期延长至20天,显著优于自由Volasertib(*p)。相比之下,2%和4% Tf靶向密度的TPVol展现出了优越的抑制AML病程的能力。在11天给药结束时,AML细胞仅有极少的扩增,最终将中位生存期延长至27天,显著优于无靶向组(*p)。然而6% Tf靶向密度的TPVol却表现出了弱于2%和4%组别的抗肿瘤能力,中位生存期24天,治疗效果变差。以上结果证明,TfR靶向的囊泡能够显著提升Volasertib对AML的治疗能力,体现出了囊泡药物长循环和靶向能力的优势。
在实验中发现,在第11天停止给药后,第14天的成像结果表明AML有着明显的复发(图4 B&C),这可能是由于较低的给药剂量和较短的给药时间。选用上述组别中效果最佳的4% Tf靶向密度的TPVol实施了进一步的实验,确定了12 mg Vol/kg,4天一针共4针和9 mgVol/kg,3天一针共4针两种给药方案,在单位时间内药物剂量不变的前提下增加总给药剂量,并延长给药时间。结果表明,12 mg Vol/kg的剂量让小鼠在注射5天后体重迅速下降并死亡,但是9 mgVol/kg的剂量展现出了明显更优秀的抗肿瘤效果和中位生存期的延长。与PBS组相比,高剂量的TPVol组不仅使全身的生物发光强度减少了2个数量级(****p),与正常剂量的TPVol相比,生物发光强度也下降了3倍,且没有对小鼠产生明显的毒性,最终将中位生存期延长至37天,显著高于正常剂量的27天(*p)。现有技术中,Darren等人对荷AML小鼠持续进行重复多次的DD8/9抑制剂给药,并最终延长了中位生存期至24.5天,Kavitha等人通过连续7次的模拟肽MYB: CBP抑制剂给药治疗AML小鼠,最终延长了17天的中位生存期。以上动物实验的结果表明,Tf-CPs能够高效并稳定地装载Volasertib,并展现出对MV-4-11细胞良好的靶向能力,并且针对血液中和归巢至骨髓中的MV-4-11细胞高效地杀伤,从而表现出了显著的治疗效果和生存期的延长。
实施例五 流式细胞仪分析、组织学分析和μCT实验
AML小鼠按上述方法用分别用PBS,Volasertib,PVol,4% TPVol(6 mgVolasertib/kg)和4% TPVol(9 mg Volasertib/kg)治疗(n=3),在第14天处死小鼠并解剖,收集小鼠的外周血(peripheral blood,PB),主要脏器,后腿股骨和胫骨,用于AML细胞浸润的流式分析和组织学分析。
为了测定AML细胞的浸润情况,收集PB,将部分的肝脏、脾脏和后腿骨研磨得到细胞悬液。将细胞离心(600 g,5 min),并在4℃下用5 mL ACK红细胞裂解液处理5 min,随后加入10 mL PBS终止,离心(600 g,5 min)获得细胞。根据抗体制造商的流式细胞分析方案,进一步使用CD45-APC抗体标记细胞并用于流式检测。此外,根据前文所述,将从骨髓中收集到的细胞进一步用Western blot进行PLK1水平的检测。对后腿股骨和胫骨进行μCT测试(SkyScan 1176,Belguim)并用NRecon软件进行分析。第14天时,取主要脏器、后腿股骨、胫骨用10%福尔马林固定,石蜡包埋切片并进行组织学分析:用苏木精-伊红染色(H&E)显示不通器官的白血病细胞浸润;采用TRAP染色试剂盒(Servicebio,湖北)对胫骨和股骨的破骨细胞进行TRAP染色。用Olympus BX41显微镜观察H&E和TRAP染色切片。
为了进一步研究治疗后小鼠体内残留的AML细胞的浸润情况,在第14天时将相同方法建模并治疗的小鼠处死,取肝脏,脾脏,骨髓(Bone Marrow,BM)和外周血(PB)获得细胞并用流式细胞仪分析AML细胞浸润情况。流式的结果表明,PBS组在骨髓(76.4%),肝脏(36.7%)和外周血(9.1%)中均有明显的白血病细胞浸润,在脾脏(2.5%)中检测到较少浸润的白血病细胞(图5 A)。而经过自由Volasertib或其制剂治疗过的小鼠,检测到了明显减少的白血病细胞浸润。其中,4% TPVol治疗组中肝脏、脾脏、骨髓和外周血中白血病细胞的比例分别为3.0%、0.7%、1.2%和1.3%,显著低于PVol组(5.7%、1.2%、2.2%和1.6%)和自由Volasertib组(11.6%、1.9%、7.7%和4.8%)。令人兴奋的是4% TPVol在9 mg Vol/kg剂量的治疗后,在肝脏、脾脏、骨髓和外周血中几乎检测不到AML细胞(0.3%,0.2%,0.2%和0.2%)(图5B)。根据之前的研究,AML细胞可以重塑骨髓腔内,使其成为有利于白血病细胞生长和抑制正常造血的环境,从而促进AML细胞的增殖。而高剂量的TPVol能够显著降低,几乎完全抑制了小鼠骨髓内的白血病细胞(99.8%)。而抗体和抑制剂治疗白血病后,通常在骨髓中依然可见白血病细胞,对比之下TPVol具有更卓越的治疗效果。
在第14天从PBS、自由Volasertib、PVol,4% TPVol和4% TPVol高剂量共5组小鼠中每组随机挑选一只小鼠解剖,并将主要脏器和后腿骨进行切片并用H&E染色。图片显示PBS组的肝脏和骨髓中可见大量白血病细胞浸润,脾脏红髓明显出现坏死(图6 A)。Volasertib,PVol和4% TPVol组的白血病细胞浸润明显减少,高剂量组中几乎没有可见的白血病细胞浸润。而心脏、肺和肾脏的切片中,所有组别均没有显示出明显的浸润或病变情况。对第14天收集到的骨髓细胞中PLK1水平进行了测试,可以发现无论是TPVol正常剂量组还是高剂量组,均呈现明显更低的PLK1和p-Akt表达(图6 B&C)。以上结果证明,TPVol能够有效地抑制AML细胞在脏器和骨髓中浸润,验证了其出色生物相容性,以及卓越的抗AML能力。
上述的结果已经证明TPVol能够有效治疗原位AML小鼠,而相关研究表明,AML通常伴随着骨损伤的发生。骨保护素和NF-κB受体激活剂配体(Receptor Activator of NF-κBLigand, RANKL)主要调控骨形成和骨吸收,其可刺激破骨细胞活化,导致骨损伤。为了评估骨髓中微环境中的损伤,进行了酒石酸耐酸性磷酸酶(TRAP)染色和微计算机断层扫描(μCT)分析,以评估破骨细胞的活性和骨吸收。TRAP染色图像(图7 A)显示PBS组小鼠的股骨和胫骨表面有大量染成红色的破骨细胞,而TPVol极大地减少了破骨细胞的数量。此外,在TPVol高剂量组中发现的破骨细胞较少,说明骨髓中中AML细胞的扩张受到强烈抑制。μCT图像显示PBS组中骨小梁结构明显缺失(图7 B),而TPVol和高剂量组的骨结构与健康小鼠接近。同时μCT定量分析显示,PBS组的小鼠的骨小梁数量(Tb.N)、骨体积分数(BV/TV)和骨表面分数(BS/BV)降低,骨小梁间距(Tb.sp)增加(图7 C)。而与健康小鼠相比,TPVol高剂量组的小梁组织和骨密度几乎完整,由于骨髓中白血病细胞浸润极少,因此破骨细胞的产生也很少。以上结果均表明,TPVol在体内外对AML均表现出了非凡的靶向性、安全性和治疗效果,对AML造成的骨损伤具有很好的预防和保护作用。
对比例 A6-CPs-Vol
A6-PEG-P(TMC-DTC)和PEG-P(TMC-DTC)-PAsp(A6摩尔含量为30%)溶解在DMSO中(40 mg/mL)。取25 μL聚合物溶液与小分子药物Volasertib混合均匀后打入到持续搅拌的0.975 mL Hepes缓冲溶液(pH 6.8,5 mM),然后在室温下静置2小时后,用PB透析8小时(MWCO 14000 Da),该过程中聚合物囊泡自交联,得到A6-CPs-Vol,粒径54.4±2.1nm,PDI为0.18±0.01,DLE为51.5%,DLC4.9 wt.%,理论载药量9.1 wt.%。
参考实施例二的细胞毒性实验,测试了A6-CPs-Vol对人源急性髓系白血病细胞株MV-4-11的毒性,A6-CPs-Vol的IC50为41.0 nM。
接种了MV-4-11-Luc-GFP的小鼠被随机分为4组,每组6只(n=6):PBS,Volasertib,高剂量Volasertib和A6-CPs-Vol,在第5,8,11和14天时尾静脉给药(7.5 mg Volasertib/kg,高剂量25 mg Volasertib/kg。在第5,11和14天腹腔注射D-荧光素钾盐(50 mg/kg),在注射的10分钟后用异氟烷进行吸入式麻醉并使用IVIS成像系统检测生物发光情况,用LivingImage 2.6软件进行分析。每2天检测小鼠体重并观察生存期至第22天,绘制Kaplan-Meier生存曲线。进行了动物实验来探究A6-CPs-Vol能否提高Volasertib对MV-4-11的治疗能力,从图8体重和生存期中可以看出,高剂量的Volasertib组以及正常剂量的A6-CPs-Vol组都发生了明显的体重降低和提前死亡,A6-CPs-Vol产生了预料之外的现象,产生了严重的毒副作用。
本发明中,转铁蛋白功能化的具有不对称膜结构的囊泡Tf-CPs能够利用带负电的聚天冬氨酸内核有效装载Volasertib(TPVol)并能够体内靶向递送至MV-4-11 AML细胞并实现治疗(图9)。本发明制备了TfR靶向的装载有PLK1抑制剂Volasertib的囊泡TPVol,载药量达到5.1wt.%,粒径在48-51 nm之间,具有出色的稳定性和还原响应性。在细胞层面验证了TPVol能够靶向并高效杀伤TfR过表达的MV-4-11细胞。在动物层面上首先验证了TPVol能够在体内长循环并最终靶向富集到归巢至骨髓的MV-4-11细胞,随即在治疗AML小鼠的实验中证实通过尾静脉注射TPVol能够有效抑制AML细胞增殖,显著减少AML细胞在器官内的浸润,延长AML小鼠中位生存期,并保护骨结构不被破坏。与自由Volasertib相比,TPVol具有更高的细胞毒性,PLK1抑制能力和体内的治疗效果,这主要是由于囊泡药物具有粒径有利于细胞摄取,循环稳定性高,生物相容性好,靶向性强等优点。以聚合物囊泡作为载体,开发靶向制剂,提高临床药物的治疗效果,是未来改善临床药物使用的有效尝试。
Claims (6)
1.一种转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡,包括聚合物囊泡;所述聚合物囊泡内装载PLK1抑制剂,其特征在于,所述聚合物囊泡外接有转铁蛋白;所述聚合物囊泡中,聚合物的侧链含有双硫键;PLK1抑制剂为Volasertib;聚合物为N3-PEG-P(A-DTC)与PEG-P(A-DTC)-PAsp,A为TMC、LA或者CL;DTC的化学结构式如下:
。
2.根据权利要求1所述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡,其特征在于,N3-PEG-P(A-DTC)的摩尔量为N3-PEG-P(A-DTC)与PEG-P(A-DTC)-PAsp总摩尔量的2~15%。
3.根据权利要求1所述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡,其特征在于,聚合物为两亲性聚合物,其中,亲水链段的分子量为3~15 kg/mol,疏水链段的分子量为10~30kg/mol。
4.权利要求1所述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡的制备方法,其特征在于,将聚合物囊泡装载PLK1抑制剂,然后修饰转铁蛋白,得到转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡。
5.根据权利要求4所述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡的制备方法,其特征在于,将PLK1抑制剂、聚合物混合、透析,得到装载PLK1抑制剂的聚合物囊泡,然后将装载PLK1抑制剂的聚合物囊泡与带有蛋白点击试剂的转铁蛋白反应,得到转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡。
6.权利要求1所述转铁蛋白受体靶向的装载PLK1抑制剂的聚合物囊泡在制备抗急性髓系白血病药物中的应用。
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