CN106798923B - 功能靶向性载体材料二硬脂酰磷脂酰乙醇胺-聚乙二醇-聚乙烯亚胺化合物及其修饰的脂质体 - Google Patents
功能靶向性载体材料二硬脂酰磷脂酰乙醇胺-聚乙二醇-聚乙烯亚胺化合物及其修饰的脂质体 Download PDFInfo
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Abstract
本发明公开了一种功能靶向性载体材料二硬脂酰磷脂酰乙醇胺‑聚乙二醇‑聚乙烯亚胺化合物及其修饰的脂质体。本发明将DSPE‑PEG2000‑PEI600化合物作为靶向性分子修饰于载药脂质体表面,能够使药物有效通过血脑屏障,选择性聚集于肿瘤细胞,产生靶向作用。比如,使C6脑胶质瘤细胞及脑胶质瘤干细胞对药物的摄取比率显著提高,在C6脑胶质瘤细胞和脑胶质瘤干细胞的线粒体上使药物有最强的选择性集中分布,使药物对脑胶质瘤细胞表现为更强的生长抑制作用,使药物显示出更强的跨越血脑屏障、杀伤脑胶质瘤干细胞的效应,即双重靶向性作用,可延长药物在ICR小鼠体内的循环时间和在肿瘤组织中的聚集程度,从而延长生物的生存期。
Description
技术领域
本发明属于药物领域,涉及一种功能靶向性载体材料二硬脂酰磷脂酰乙醇胺-聚乙二醇-聚乙烯亚胺(DSPE-PEG2000-PEI600)化合物及其修饰的脂质体。
背景技术
脑肿瘤是目前威胁人类健康的重大疾病之一。脑胶质瘤作为一种恶性的脑肿瘤,其难治性主要表现在如下几个方面:脑胶质瘤特殊的发病位置及其浸润性生长的特点使得手术治疗无法将其彻底切除干净;通过放射治疗方法治疗脑胶质瘤副作用大,耐受性较差;传统的化疗药物无法穿透血脑屏障达到脑胶质瘤区域;综合治疗后残存的脑胶质瘤干细胞容易导致肿瘤复发。如何使化疗药物跨越血脑屏障、清除残存的脑胶质瘤及其干细胞瘤是亟待解决的科学问题。
聚乙烯亚胺(PEI)是一种阳离子聚合物,其阳离子的特性可以帮助有效包裹和压缩负电性的DNA分子,因此在实验研究中常将其用作质粒DNA、siRNA等的递送载体。然而,大分子的PEI对人体内的正常组织有较强的细胞毒性,使得其在体内的应用受到严重制约。研究发现,经修饰的小分子PEI表现为较低的细胞毒性,可用于提升载体在负电性的细胞和亚细胞结构中的摄取。本发明中采用DSPE-PEG2000脂质材料修饰小分子PEI(PEI600),合成功能性材料DSPE-PEG2000-PEI600,并应用于载药脂质体,使可以通过电荷相互作用经吸附介导内吞作用机制跨越血脑屏障,靶向脑胶质瘤干细胞,并选择性的聚积于肿瘤细胞的线粒体内。
二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG2000)是一种用于长循环脂质体制备的材料。1992年Maruyama等就报导了将DSPE-PEG2000材料用于大单层脂质体的制备,可以显著增加药物在血液系统中循环时间的实验结果。由于长链PEG的存在,修饰有二硬脂酰基磷脂酰乙醇胺-聚乙二醇材料的脂质体可以有效避免人体内网状内皮系统(RES)对脂质体的清除,从而提高载药系统在血液循环系统中的稳定性,使其具有生物学稳定的性质,延长脂质体在体内的循环时间。并且,该种具有适当粒径的长循环脂质体可以在肿瘤组织的非正常血管区域表现出渗透与滞留增强效应(enhanced permeability and retentioneffect,EPR effect),可以实现肿瘤部位的被动靶向,从而增加药物在肿瘤部位的聚集。
发明内容
本发明的目的是提供一种功能靶向性载体材料二硬脂酰磷脂酰乙醇胺-聚乙二醇-聚乙烯亚胺(DSPE-PEG2000-PEI600)化合物及其修饰的脂质体。
其中,所述重复结构单元a为-NHCH2CH2-,总个数为x,x为0-15;
重复结构单元b为-N(CH2CH2NH2)CH2CH2-,总个数为y,y为1-10。
上述DSPE-PEG2000-PEI600化合物中,重复结构单元a和b的连接方式不固定,可为各种连接方式。
本发明提供的制备上述化合物的方法,包括如下步骤:将PEI600和式III所示化合物(DSPE-PEG2000-NHS)于溶剂中进行取代反应,反应完毕得到所述聚合物DSPE-PEG2000-PEI600化合物;
所述PEI600为以-NH2和-CH2CH2NH2为端基,由重复结构单元a和重复结构单元b构成的聚合物;
其中,所述重复结构单元a为-NHCH2CH2-,总个数为x,x为0-15;
重复结构单元b为-N(CH2CH2NH2)CH2CH2-,总个数为y,y为1-10;
所述重复结构单元a和b的连接方式不固定,可为各种连接方式。
具体的,所述PEI600可为由以-NH2和-CH2CH2NH2为端基,依次由重复结构单元a和重复结构单元b相连构成的聚合物;
上述方法中,所述溶剂选自DMF、DMSO和氯仿中的至少一种;
所述PEI600和式III所示化合物的投料摩尔比为1:0.5-2,优选为1:1;
所述PEI600与所述溶剂的用量比为20μmol:1-10mL,优选为20μmol:4mL;
所述取代反应步骤中,温度通常为室温,时间为24h-48h,优选为24h;该取代反应是由PEI分子上的氨基亲核取代DSPE-PEG2000-NHS上的NHS基团形成酰胺键而得式I;
所述取代反应在惰性气氛中进行;所述惰性气氛如氩气气氛。
所述方法还包括如下步骤:在所述反应完毕后,将所得反应体系置于截留分子量为2500Da左右的透析袋中用水进行透析;
所述透析步骤中,时间通常为36h-72h,优选为48h。
所述方法还包括如下步骤:在所述用水进行透析步骤之后,将透析所得液体进行冻干的步骤。经过该冻干步骤,可得到干燥的呈现白色粉末状的聚合物DSPE-PEG2000-PEI600化合物。
本发明的再一个目的是提供聚合物DSPE-PEG2000-PEI600化合物在制备靶向性产品中的应用。
上述应用中,所述靶向性产品为靶向性药物产品,具体可为靶向性药物载体,更具体可为靶向性脂质体,再具体可为靶向性空白脂质体、靶向性柔红霉素脂质体、靶向性香豆素脂质体或靶向性DiR脂质体。
本发明还提供了一种制备PEI修饰的靶向性空白脂质体的方法,该方法为以卵磷脂、胆固醇、前述本发明提供的聚合物DSPE-PEG2000-PEI600化合物和DSPE-PEG2000为原料制得;
该方法具体包括如下步骤:
1)将卵磷脂、胆固醇、前述本发明提供的聚合物DSPE-PEG2000-PEI600化合物和DSPE-PEG2000于有机溶剂中溶解后,旋转蒸发减压干燥除去所述有机溶剂,得到脂膜;
所述DSPE-PEG2000的结构式如下:
2)向步骤1)所得脂膜中加入硫酸铵水溶液,进行水浴超声5min后,再在超声波细胞粉碎机进行超声,将超声所得含有粗脂质体的液体通过孔径为400nm的聚碳酸酯膜3次后,再通过孔径为200nm的聚碳酸酯膜3次后,于透析袋中透析,得到所述PEI修饰的靶向性脂质体。
上述方法的步骤1)中,有机溶剂选自氯仿、二氯甲烷和由二氯甲烷与甲醇组成的混合液中的至少一种;
所述卵磷脂的投料摩尔份数为60-65份;所述胆固醇的投料摩尔份数为30-35份;所述聚合物DSPE-PEG2000-PEI600化合物和DSPE-PEG2000的投料摩尔份数均为0.5-5份;
具体的,卵磷脂、胆固醇、述聚合物DSPE-PEG2000-PEI600化合物和DSPE-PEG2000的投料摩尔比为63:32.5:0.5:4;
所述步骤2)中,硫酸铵水溶液的浓度为200-300mM,具体为250mM;
所述水浴超声步骤中,超声的能量为100-200W;温度为20-30℃;
所述在超声波细胞粉碎机进行超声的步骤中,超声的能量为100-300W,具体为200W;温度为20-40℃,具体为35℃;超声工作时间为10s,间歇时间为10s,全程时间为10min;
所述透析步骤中,透析袋的截留分子量为10,000-12,000Da;透析的时间为12-48小时,具体为24小时;在实际操作中,该透析步骤可每8小时更换一次透析液,如总透析时间为24小时,可总共更换3次透析液,以达到更好的透析效果。
透析所用试剂为HBS缓冲溶液;
所述HBS缓冲液的成分如下:151mMNaCl、25.2mMHepes,PBS pH值为7.4。
另外,按照上述方法制备得到的PEI修饰的靶向性空白脂质体,也属于本发明的保护范围。
本发明还提供了一种PEI修饰的靶向性柔红霉素脂质体,该PEI修饰的靶向性柔红霉素脂质体是以本发明PEI修饰的靶向性空白脂质体和柔红霉素为原料制得。
上述PEI修饰的靶向性柔红霉素脂质体中,所述靶向性柔红霉素脂质体的包封率大于95%。
本发明还提供了一种制备上述PEI修饰的靶向性柔红霉素脂质体的方法,该方法包括如下步骤:
将本发明PEI修饰的靶向性空白脂质体和盐酸柔红霉素的水溶液于水浴中振荡,得到所述PEI修饰的靶向性柔红霉素脂质体。
上述方法中,所述盐酸柔红霉素和所述PEI修饰的靶向性空白脂质体的质量比为1:10-100,具体为1:20;
所述振荡步骤中,温度为35-70℃,具体为60℃;
时间为10-45min,具体为20min。
此外,上述本发明提供的PEI修饰的靶向性柔红霉素脂质体在制备真核生物肿瘤细胞增殖的抑制剂、制备真核生物肿瘤细胞凋亡诱导剂、制备真核生物肿瘤细胞内caspase8和/或caspase 3活化诱导剂和提升真核生物肿瘤细胞的摄取中任意一种的应用以及在制备预防和/或治疗肿瘤的药物中的应用,也属于本发明的保护范围。其中,所述真核生物具体为哺乳动物;所述肿瘤细胞具体为癌细胞;所述癌细胞具体为脑胶质瘤细胞,更具体为鼠源C6细胞或脑胶质瘤干细胞;
所述肿瘤具体为脑胶质瘤,更具体为脑胶质瘤干细胞。
本发明将DSPE-PEG2000-PEI600化合物作为靶向性分子修饰于载药脂质体表面,能够使药物有效通过血脑屏障,选择性聚集于肿瘤细胞,产生靶向作用。比如,使C6脑胶质瘤细胞及脑胶质瘤干细胞对药物的摄取比率显著提高,在C6脑胶质瘤细胞和脑胶质瘤干细胞的线粒体上使药物有最强的选择性集中分布,使药物对脑胶质瘤细胞表现为更强的生长抑制作用,使药物显示出更强的跨越血脑屏障、杀伤脑胶质瘤干细胞的效应,即双重靶向性作用,可延长药物在ICR小鼠体内的循环时间和在肿瘤组织中的聚集程度,从而延长生物的生存期。本发明的这些特性使得本发明较现有发明具有优越性。
附图说明
图1为式I所示化合物DSPE-PEG2000-PEI600的合成路线。
图2为PEI600(A)),DSPE-PEG2000-NHS(B))和反应产物DSPE-PEG2000-NHS和DSPE-PEG2000-PEI600的混合物(C))的MALDI-TOF质谱图。
图3为PEI修饰的靶向性脂质体的粒径分布情况。
图4为载药脂质体药物的累计释放率(%)),释放介质为含有10%胎牛血清的pH为7.4的PBS溶液。
图5为柔红霉素脂质体组(b))和PEI修饰的靶向性柔红霉素脂质体组(c))在脑胶质瘤干细胞(A))、C6脑胶质瘤细胞(B))中的药物摄取情况。
图6为空白对照(a),柔红霉素脂质体(b),PEI修饰的靶向性柔红霉素脂质体(c)在脑胶质瘤干细胞(A)和C6脑胶质瘤细胞(B)中的线粒体共定位情况,其中细胞的线粒体用线粒体红色荧光探针标记。
图7为柔红霉素脂质体和PEI修饰的靶向性柔红霉素脂质体对脑胶质瘤干细胞的细胞毒效应。
图8为体外血脑屏障模型的示意图(A);柔红霉素脂质体(a)和PEI修饰的靶向性柔红霉素脂质体(b)给药后,药物跨越血脑屏障(BBB)并杀伤脑胶质瘤细胞及脑胶质瘤干细胞的情况(B)。
图9为各个制剂组给药后对脑胶质瘤干细胞中凋亡信号通路中凋亡蛋白和凋亡酶的调节情况;其中(A)脑胶质瘤干细胞内Caspase 3,Caspase 8和Bax在高内涵系统下的表达情况的荧光图像;(B)Caspase 3的活性比率;(C)Caspase 8的活性比率;(D)Bax的活性比率;(E)Mcl 1的活性比率;其中1.为空白对照组;2.为柔红霉素脂质体组;3.为PEI修饰的靶向性柔红霉素脂质体组;以上结果均由Operetta高内涵筛选系统测定并由Columbus系统分析。
图10为PEI修饰的靶向性脂质体在脑胶质瘤荷瘤小鼠体内的分布情况。注:(A)经尾静脉给药后,各个制剂组在脑胶质瘤荷瘤小鼠体内的实时分布情况图像;(B)在ICR小鼠给药48h后,解剖出心、肝、脾、肺、肾和荷瘤脑组织的体外成像图像;其中a.为空白对照;b.为DiR脂质体;c.为PEI修饰的靶向性DiR脂质体。
图11为脑胶质瘤荷瘤小鼠在肿瘤接种并接受各个制剂组治疗后的卡普兰-迈耶生存曲线。
具体实施方式
下面结合具体实施例对本发明作进一步阐述,但本发明并不限于以下实施例。
所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
其中,实施例所用材料的来源如下:
二硬脂酰基磷脂酰乙醇胺-聚乙二醇-N-琥珀酰亚胺(1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-polyethyleneglycol2000-N-hydroxysuccin-imide,DSPE-PEG2000-NHS),购自日本油脂株式会社(日本NOF公司),产品目录号为M139522。
聚乙烯亚胺600(Polyethylenimine,PEI600),购自Alfa Aesar阿法埃莎(中国)化学有限公司,产品目录号为G18Y027。
N,N-二甲基甲酰胺(Dimethylformamide,DMF),购自美国Acros Organics公司,产品目录号为1339870。
蛋黄卵磷脂(EPC)购自日本油脂株式会社,产品目录号为108057-3。
胆固醇购自北京市海淀区微生物培养基制品厂,批号20020106。
DSPE-PEG2000购自美国Avanti Polar Lipids公司,产品目录号880120P;
聚碳酸酯膜购自美国Millipore公司,产品编号为HTTP02500;
实施例1、式I所示功能靶向性载体材料DSPE-PEG2000-PEI600的合成与表征
将20μmolPEI600(x为0-15,y为1-10)和20μmol式III所示化合物DSPE-PEG2000-NHS溶解在4ml无水DMF中,将反应液混合物在室温、氩气保护下用磁力搅拌器轻轻搅拌24h进行取代反应,反应完毕得到粗产物,继而将粗产物转移到再生纤维素透析袋(截留分子量2500),在去离子水中透析48h,除去未反应的PEI和DMF溶剂。接下来将反应液冻干,得到干燥白色粉末式I所示产物,在-20℃条件下保存。
该产物的合成路线如图1所示。
反应产物采用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)验证反应目标产物的存在。MALDI-TOF-MS使用2,5-二羟基苯甲酸(DHB)为基体。
图2分别为原料PEI600、DSPE-PEG2000-NHS和产物DSPE-PEG2000-PEI600的MALDI-TOF-MS图谱。如图2所示,反应产物中DSPE-PEG2000-PEI600的平均分子质量为3507.3Da(图2C),PEI600的平均分子质量为534.5Da(图2A),DSPE-PEG2000-NHS的平均分子质量为3065.3Da(图2B)。DSPE-PEG2000-PEI600分子与PEI600分子之间的质量差,与脱去一分子NHS后的DSPE-PEG2000-NHS分子质量相吻合。可见,所得产物结构正确,为目标产物,且x为0-15;y为1-10。
实施例2、脂质体的制备与表征
1)PEI修饰的靶向性空白脂质体的制备
a、精密称取卵磷脂(EPC)、胆固醇(CHOL)、DSPE-PEG2000-PEI600、DSPE-PEG2000按摩尔比63:32.5:0.5:4于茄形瓶中,加入适量氯仿溶解,然后在40℃水浴、40rpm转速下通过旋转蒸发减压干燥除去有机试剂,在茄形瓶底部及内壁形成一层薄薄的均匀脂膜;
b、向步骤1)所得脂膜中加入适量250mM硫酸铵溶液水化:先在水浴中于室温超声5min,超声能量为100W,至形成乳白色均匀的粗脂质体后转移到JY92-IID型超声波细胞粉碎机中进一步超声(设置超声工作时间为10s,间歇时间为10s,全程时间为10min,保护温度为35℃,功率为200W)。超声结束后粗脂质体逐渐形成带有微弱蓝色荧光的半透明液体,然后将其通过孔径为400nm的聚碳酸酯膜3次后,再通过孔径为200nm的聚碳酸酯膜3次后后,将脂质体混悬液装入透析袋(截留分子量10,000-12,000Da),在HBS缓冲溶液中(151mMNaCl,25.2mMHepes,PBS pH 7.4)透析24小时,每8小时更换一次透析液,共三次,得到PEI修饰的靶向性空白脂质体。
2)PEI修饰的靶向性柔红霉素脂质体的制备
采用硫酸铵梯度法,将柔红霉素包载入步骤1)所得靶向性空白脂质体内,于水相中制得PEI修饰的靶向性柔红霉素脂质体。
具体步骤包括:
将盐酸柔红霉素以一定的浓度溶解在蒸馏水中后,将该盐酸柔红霉素的水溶液与步骤1)所得PEI修饰的靶向性空白脂质体分别在60℃水浴中预热,然后按照药脂比(也即盐酸柔红霉素和PEI修饰的靶向性空白脂质体的质量比)1:20,将该盐酸柔红霉素的水溶液加入到步骤1)所得PEI修饰的靶向性空白脂质体的水溶液中,在60℃空气浴恒温培养振荡器里振摇20min,得到本发明提供的PEI修饰的靶向性柔红霉素脂质体。
实施例2所用柔红霉素脂质体可按照如下方法制备而得:
与PEI修饰的靶向性柔红霉素脂质体的制备方法相同,但在成膜过程中将靶向性载体材料DSPE-PEG2000-PEI600替换为等摩尔量的DSPE-PEG2000。
3)脂质体的表征
取步骤1)所得PEI修饰的靶向性空白脂质体500μl通过Sephadex G-50葡聚糖凝胶柱,以HBS缓冲溶液(151mMNaCl,25.2mMHepes,PBS pH 7.4)为流动相预饱和葡聚糖凝胶柱。
之后取载药脂质体(柔红霉素脂质体、步骤2)所得PEI修饰的靶向性柔红霉素脂质体)500μl通过Sephadex G-50葡聚糖凝胶柱,以HBS缓冲溶液为流动相分离未包进脂质体的游离柔红霉素,收集分离后的脂质体,加入九倍体积甲醇破坏,并用流动相稀释后,用上述高效液相色谱法(HPLC)进行测定。
取未过凝胶柱的柔红霉素脂质体、步骤2)所得PEI修饰的靶向性柔红霉素脂质体原液,加入九倍体积甲醇破坏,并用流动相稀释相同倍数后,用上述高效液相色谱法进行测定。
采用下面的公式对柔红霉素的包封率进行计算:
包封率(%)=过凝胶柱分离后脂质体中的药物含量/未过凝胶柱脂质体中的药物含量×100%。
药物浓度用对照品法(比较一点法)计算,即得。
分别取新制得的脂质体加入PBS稀释载药脂质体至1ml,混合均匀后,使用NanoSeries Zen 4003ZetaSizer(Malvern instruments,Ltd,UK)测定脂质体的粒径、多分散度和Zeta电位。
将柔红霉素脂质体和步骤2)所得PEI修饰的靶向性柔红霉素脂质体在含血清蛋白的释放介质(含10%胎牛血清的PBS缓冲液)中进行体外释放实验。
具体步骤包括:取2ml脂质体,加入2ml释放介质混匀后放入透析袋(分子截留量为10,000-12,000Da)内,两端扎紧后将透析袋置于10.0ml释放介质中,在37℃、100rpm的条件下在摇床上振荡。分别于0,0.25,0.5,1,2,4,6和24h时取出0.2ml释放介质,并每次取样后立即补入同等体积的新释放介质。取出的各份样品用九倍体积甲醇进行破坏稀释,使蛋白溶解,高速离心,再用滤膜过膜,除去大分子蛋白,用HPLC进行检测,测定各样品中各样品的峰面积,用其线性回归曲线换算得到相应浓度。然后计算出各个样品在不同时刻的释放量,进而得到其在某一时刻的累积释放量。
各种脂质体的体外释放率分别用下列公式计算:
体外释放率(%)=第i时间点释放液中药物的量/与透析体积相同的透析前脂质体溶液中药物的量×100%。
(4)脂质体的表征结果
柔红霉素脂质体和步骤2)所得PEI修饰的靶向性柔红霉素脂质体的药物包封率、粒径、多分散系数和Zeta电位的表征结果如表1所示。
表1、脂质体的包封率、粒径及Zeta电位表征
数据为平均值±标准差的形式(n=3)。
结果显示,上述脂质体的平均粒径在100nm左右,分布均一。柔红霉素在两种脂质体中的包封率均大于95%。两种脂质体的Zeta电位均呈负电性。
图3为步骤2)所得PEI修饰的柔红霉素脂质体的粒径分布情况,可见该脂质体粒径100nm左右,粒径均一,分散度良好。
图4为柔红霉素从步骤2)所得PEI修饰的靶向性柔红霉素脂质体和柔红霉素脂质体的体外释放率结果。选择含10%胎牛血清的PBS缓冲液作为释放介质是为了模拟动物体内的血液环境,更加客观地模拟载药脂质体经静脉注射后在血液循环过程中的释放。实验结果表明,柔红霉素从各种脂质体中的释放率,在最初的2h内,释放率均低于5%,在第24h,步骤2)所得PEI修饰的靶向性柔红霉素脂质体和柔红霉素脂质体的体外累积释放率分别为7.49±2.44%及9.87±3.04%。
实施例3、PEI修饰的柔红霉素脂质体的药效实验
(1)细胞摄取情况
图5所示为脑胶质瘤干细胞(A)、C6脑胶质瘤细胞(B)在各个制剂组给药处理后细胞摄取的荧光强度结果。给药组分别为柔红霉素脂质体组(b)和PEI修饰的柔红霉素脂质体组(c)。相应的数值在柱形图中体现。
给药孵育4h后,柔红霉素脂质体组、实施例1步骤2)所得PEI修饰的柔红霉素脂质体组在脑胶质瘤干细胞内的摄取比率分别为1.00±0.03、2.18±0.17;在C6脑胶质瘤细胞内的摄取比率分别为1.00±0.01、2.28±0.11。
结果显示,C6脑胶质瘤细胞及脑胶质瘤干细胞对实施例1步骤2)所得PEI修饰的柔红霉素脂质体组的摄取比率显著高于柔红霉素脂质体组。
(2)线粒体靶向性效应
图6为各制剂组在C6脑胶质瘤细胞及脑胶质瘤干细胞中的亚细胞定位激光共聚焦显微图像。如图所示,细胞线粒体通过线粒体红荧光探针染成红色,各个柔红霉素制剂组呈现绿色的荧光。在荧光叠加的图中,黄色荧光为绿色和红色荧光的组合,表示制剂与线粒体的共定位。
结果显示,实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体组表现为最明显的黄色荧光,而柔红霉素脂质体组中则没有显示黄色荧光。说明这两个制剂组在C6脑胶质瘤细胞和脑胶质瘤干细胞的线粒体上有最强的选择性集中分布。
(3)对脑胶质瘤细胞及脑胶质瘤干细胞的抑制效应
图7所示为柔红霉素脂质体和实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体对脑胶质瘤干细胞的抑制作用。由图可知,实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体与柔红霉素脂质体相比,对脑胶质瘤细胞表现为更强的生长抑制作用。
(4)体外跨越血脑屏障后对脑胶质瘤干细胞的杀伤效应
建立BMVEC/脑胶质瘤干细胞共培养模型,如图8A所示,并用于实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体的体外跨越血脑屏障后对脑胶质瘤干细胞的杀伤效应(双重靶向性效应)评价。柔红霉素脂质体和实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体在跨越血脑屏障后对脑胶质瘤干细胞的生长抑制情况见图8B。
结果显示,脑胶质瘤干细胞的存活率分别为:实施例1步骤2)所得PEI修饰的柔红霉素脂质体(70.62±0.98%),以及柔红霉素脂质体(72.96±2.78%)。实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体,与柔红霉素脂质体制剂组相比,显示出更强的跨越血脑屏障、杀伤脑胶质瘤干细胞的效应,即双重靶向性作用。
(5)对脑胶质瘤干细胞的诱导凋亡效应与机制
图9所示为脑胶质瘤干细胞在给以柔红霉素脂质体和PEI修饰的靶向性柔红霉素脂质体6小时后,采用高内涵筛选系统对细胞内凋亡蛋白Caspase 3、Caspase 8和Bax的表达情况进行测定的荧光图谱。图中绿色荧光的强度代表细胞内凋亡蛋白表达的量。
结果显示,凋亡蛋白酶Caspase 3、Caspase 8和促凋亡蛋白Bax均在给药孵育后而呈现表达量提高的趋势,其中,实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体制剂组较柔红霉素脂质体呈现更高的表达量。
脑胶质瘤干细胞在给以柔红霉素脂质体和PEI修饰的靶向性柔红霉素脂质体6小时后,采用高内涵筛选系统对细胞内凋亡相关信号通路的蛋白Caspase 3、Caspase 8、Bax和Mcl 1的表达情况进行测定,相应的蛋白活性比率的数值以柱形图的形式体现。如图9B、C、D&E所示,空白培养基、柔红霉素脂质体、PEI修饰的靶向性柔红霉素脂质体给药后,脑胶质瘤干细胞中凋亡蛋白酶Caspase 8的活性比率分别为1.00±0.03,1.01±0.04,1.53±0.01(图9B);凋亡蛋白酶Caspase 3的活性比率分别为1.00±0.01,1.07±0.01,1.09±0.01(图9C);促凋亡蛋白Bax的活性比率分别为1.00±0.01,1.01±0.02,1.07±0.01(图9D);抗凋亡蛋白Mcl 1的活性比率分别为1.00±0.01,0.97±0.01,0.93±0.01(图9E)。
(6)在荷瘤小鼠体内的分布情况
DiR标记的功能靶向性脂质体在脑胶质瘤荷瘤ICR小鼠体内的分布和肿瘤蓄积能力如图10所示。图10A为尾静脉注射游离DiR、DiR脂质体、PEI修饰的靶向性DiR脂质体后各个时间点下ICR小鼠体内的荧光分布;图10B为尾静脉注射游离DiR、DiR脂质体、实施例1步骤2)所得PEI修饰的靶向性DiR脂质体在ICR小鼠体内48h后,解剖出心、肝、脾、肺、肾和荷瘤脑组织的体外成像结果。
从图中可以看出,尾静脉注射DiR标记的实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体后1h时,可以在ICR小鼠体内观察到强烈的DiR荧光信号,而且在肿瘤组织48h仍可观察到强烈的荧光信号。相比柔红霉素脂质体,PEI修饰的靶向性脂质体在脑肿瘤组织部位有明显的聚集。结果表明,尾静脉注射实施例1步骤2)所得PEI修饰的靶向性柔红霉素脂质体可延长药物在ICR小鼠体内的循环时间和在肿瘤组织中的聚集程度,可在肿瘤组织观察到最强的荧光信号。
(7)在荷瘤小鼠体内抗脑胶质瘤疗效评价
从肿瘤接种后第14天开始给药,柔红霉素按4.5mg/kg体重给药量,每3-4天给药1次,连续给药四次,每组7只考察生存曲线。观察脑胶质瘤荷瘤小鼠模型的行为状态,记录小鼠活动状况、症状、死亡日期,绘制卡普兰-迈耶生存曲线。结果如图11所示,脑胶质瘤荷瘤小鼠在接受生理盐水、柔红霉素脂质体、PEI修饰的靶向性柔红霉素脂质体和柔红霉素游离药的治疗后,中位生存期分别为29、32、38和27天。
Claims (20)
3.根据权利要求2所述的方法,其特征在于:所述PEI600的平均分子量为500-600Da;
所述溶剂选自DMF、DMSO和氯仿中的至少一种;
所述PEI600和式III所示化合物的投料摩尔比为1:0.5-2;
所述PEI600与所述溶剂的用量比为20μmol:1-10mL;
所述取代反应步骤中,温度为室温,时间为24h-48h;
所述取代反应在惰性气氛中进行。
4.根据权利要求3所述的方法,其特征在于:所述PEI600和式III所示化合物的投料摩尔比为1:1;
所述PEI600与所述溶剂的用量比为20μmol:4mL;
所述惰性气氛为氩气气氛。
5.权利要求1所述聚合物在制备靶向性产品中的应用。
6.根据权利要求5所述的应用,其特征在于:所述靶向性产品为靶向性药物载体。
7.根据权利要求6所述的应用,其特征在于:所述靶向性药物载体为靶向性脂质体。
8.根据权利要求7所述的应用,其特征在于:所述靶向性脂质体为靶向性空白脂质体、靶向性柔红霉素脂质体、靶向性香豆素脂质体或靶向性DiR脂质体。
9.一种制备PEI修饰的靶向性空白脂质体的方法,为以卵磷脂、胆固醇、权利要求1所述聚合物和DSPE-PEG2000为原料制备而得。
10.权利要求9所述方法得到的PEI修饰的靶向性空白脂质体。
11.一种PEI修饰的靶向性柔红霉素脂质体,为以权利要求10所述PEI修饰的靶向性空白脂质体和柔红霉素为原料制得;
所述靶向性柔红霉素脂质体的包封率大于95%。
12.一种制备权利要求10或11所述PEI修饰的靶向性柔红霉素脂质体的方法,包括如下步骤:
将权利要求10或11所述PEI修饰的靶向性空白脂质体和盐酸柔红霉素的水溶液于水浴中振荡,得到所述PEI修饰的靶向性柔红霉素脂质体。
13.权利要求10或11所述PEI修饰的靶向性柔红霉素脂质体在制备真核生物肿瘤细胞增殖的抑制剂、制备真核生物肿瘤细胞凋亡诱导剂和制备真核生物肿瘤细胞内caspase 8和/或caspase 3活化诱导剂中任意一种的应用。
14.根据权利要求13所述的应用,其特征在于:所述真核生物为哺乳动物;
所述肿瘤为脑胶质瘤。
15.根据权利要求14所述的应用,其特征在于:所述肿瘤细胞为癌细胞;
所述肿瘤为脑胶质瘤干细胞。
16.根据权利要求15所述的应用,其特征在于:所述癌细胞为脑胶质瘤细胞。
17.根据权利要求16所述的应用,其特征在于:所述脑胶质瘤细胞为鼠源C6细胞或脑胶质瘤干细胞。
18.权利要求10或11所述PEI修饰的靶向性柔红霉素脂质体在制备预防和/或治疗肿瘤的药物中的应用。
19.根据权利要求18所述的应用,其特征在于:所述肿瘤为脑胶质瘤。
20.根据权利要求19所述的应用,其特征在于:所述脑胶质瘤为脑胶质瘤干细胞。
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