CN115554239B - 一种基于奥沙利铂偶联性两亲聚合物的胶束 - Google Patents
一种基于奥沙利铂偶联性两亲聚合物的胶束 Download PDFInfo
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- CN115554239B CN115554239B CN202110742505.5A CN202110742505A CN115554239B CN 115554239 B CN115554239 B CN 115554239B CN 202110742505 A CN202110742505 A CN 202110742505A CN 115554239 B CN115554239 B CN 115554239B
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- oxaliplatin
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Classifications
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Abstract
本发明属生物技术领域,具体涉及一种基于奥沙利铂偶联性两亲聚合物的胶束。本发明利用氧化态的奥沙利铂将亲水性聚乙二醇和疏水性聚乳酸‑羟基乙酸共聚物偶联,构建新型的聚合物两亲分子,并进一步修饰维生素C类似物赋予其肿瘤靶向能力,自组装制成一种基于奥沙利铂偶联性两亲聚合物肿瘤靶向胶束,所述胶束可进一步负载疏水性探针。本发明的胶束可实现奥沙利铂的制剂化,改善体内分布,进一步提高其在肿瘤治疗的应用前景。
Description
技术领域
本发明属生物技术领域,具体涉及一种基于奥沙利铂偶联性两亲聚合物的胶束。
背景技术
据统计(CA:A Cancer Journal for Clinicians.2021,71(3):209-249.),2020年全球约有1930万例新增癌症病例和将近1000万例癌症死亡病例,其中乳腺癌、肺癌和结直肠癌位居发病率前三,而肺癌、结直肠癌和肝癌位居致死率前三。虽然基于现代医学的不断发展,人类已经拥有越来越多的方法对抗癌症,尽管在少数发达国家中癌症致死率的增长速度已有所下降(CA:A Cancer Journal for Clinicians.2020,70(1):7-30.),但在大部分发达国家及诸多的发展中国家(包括我国),癌症仍然是疾病致死的主因(LancetOncol.2020,21(7):e342-e349.)。
临床实践显示,以当前的医疗水平,癌症的首选疗法还是考虑手术治疗(Med SciMonit.2019,25:3537-3541.)。但适合手术的病例占比并不高,其原因之一为肿瘤固有的侵袭性和浸润性生长,导致普遍的术后残留和复发,甚至诱导转移(Cancer Res.2017,77(7):1548-1552.)。近年来的免疫疗法在抗肿瘤方面具有一定潜力,但对肿瘤治疗的响应度仍偏低(Nat Rev Immunol.2020,20(11):651-668.)。化学疗法基于阻断快速增值细胞的DNA复制或有丝分裂过程,抑制癌细胞的增殖、诱导癌细胞的死亡,仍是癌症治疗的中干力量(Expert Opin Pharmacother.2018,19(9):993-1001.)。
目前临床实践已使用了包括烷化剂类、铂类、蒽醌类等多种细胞毒性化疗药物,其中铂类药物无论是单剂还是多药联用,均为临床化疗方案中使用最广泛的治疗剂,使用比率达到70%左右之高(Nature Reviews Cancer.2021,21(1):37-50.)。铂类药物包括顺铂、卡铂、奥沙利铂等,通过金属Pt与两个鸟嘌呤之间的络合作用与DNA双链发生交联,形成一个封闭的五元螯合环从而阻止DNA的复制和细胞增殖,因此铂类药物在抗癌化疗方面有着活性高、抗癌作用强等优点,同时由于较少产生与其他化疗药物的交叉耐药性,铂类药物还常用于联合用药以增加疗效、降低毒性。
尽管铂类药物在抗癌方面具有诸多优势,但同时尚有若干缺陷之处,从而限制了其临床效果;其中最主要的即铂类药物在治疗的同时,伴随着若干毒副作用,例如最常见的肾毒性、多疗程导致严重过敏反应、抵抗感染的免疫力降低、以及严重的耳毒性和神经毒性等(Eur J Pharmacol.2014,740:364-378.)。此外,铂类药物一般亲水性较强,而大部分抗肿瘤药物属于疏水型药物,铂类药物与其他抗肿瘤药物联用时,往往体内命运差别巨大,难以达到体内协同抗肿瘤的目的。因此为了降低治疗风险、减少不良反应提高患者依从性、避免低剂量诱导的耐药发生等,需要药剂工作者从改善铂类药物的体内过程、组织分布、肿瘤靶向的角度出发,对其进行必要的制剂化改造,优化的模式是将铂类药物直接封装于载药系统中,可用于其他药物的共负载,从而达到减毒增效和药物共递送的目的。
生物学和医学方面现有技术中,基于纳米级微粒在生物体内流通和分布的特点,许多纳米级制剂被开发用来搭载和递送药物(尤其是抗肿瘤的化疗药),例如脂质体、胶束、纳米粒、纳米囊等(Eur J Pharm Biopharm.2015,93:52-79.)。这些纳米药物递送系统在延长血液循环时间、降低药物毒副作用方面已经表现出优良的效果,例如已上市制剂阿霉素脂质体Doxil,显著改善了阿霉素原有的心脏毒性问题。另外,在研的许多纳米递送系统更是着重关注如何将化疗药有选择性地递送至肿瘤组织,实现对肿瘤特异性的杀伤,即精准靶向治疗(Pharmacol Res.2017,115:87-95.)。利用纳米递药系统实现铂类药物制剂化,有望改善体内分布和提高药效(ACS Nano.2017,11(9):8560-8578.),但由于铂类药物本身特殊的性质(重金属类、水溶性),使得纳米制剂对其荷载的方式有限,且不易实现铂类和其他有机小分子药物的共递送。铂类药物在纳米载体的荷载主要通过金属离子Pt与有机电子供体间的螯合作用,但这种载药方式不稳定,容易被其他阴离子取代而导致药物提前泄露(ACS Applied Materials&Interfaces.2017,9(40):34603-34617.)。另外铂类药物良好的水溶性也限制了它们与大多溶解性不佳的化疗药物的共负载(Curr Med Chem.2020,27(18):3055-3078.)。
基于现有技术的基础及现状,本申请的发明人拟提供一种基于奥沙利铂偶联性两亲聚合物的胶束,以进一步提高药物在肿瘤治疗的应用前景。
发明内容
本发明的目的是基于现有技术的基础及现状,提供一种基于奥沙利铂偶联性两亲聚合物的胶束。
本发明利用氧化型奥沙利铂前药偶联亲水性聚合物聚乙二醇(PEG)和疏水性聚合物聚乳酸-羟基乙酸共聚物(PLGA),形成两亲性聚合物构筑胶束,实现负载奥沙利铂的同时,胶束的疏水核心还能包载其他疏水性小分子,本发明的实施例中以一种半花菁染料为模型疏水性小分子,并通过维生素C类似物DHAA的主动靶向,促进癌细胞对胶束的摄取,实现两种药物共同负载和可控释放。
本发明的胶束的构筑基元为两亲性的聚合物材料,所述的两亲性的聚合物材料是亲水性聚乙二醇(PEG)和疏水性聚乳酸-羟基乙酸共聚物(PLGA)偶联体;
优选地,PEG和PLGA是通过氧化态奥沙利铂进行偶联的;
优选地,PEG的平均分子量为5000,PLGA的平均分子量为4500;
优选地,该两亲性聚合物的临界胶束浓度为11.8μg/mL;
优选地,PEG-PLGA偶联体还可以进一步修饰维生素C类似物DHAA,以赋予其肿瘤靶向能力;
优选地,该胶束可实现对亲水性的抗肿瘤化疗药物奥沙利铂的有效负载,并优化其体内分布过程;
优选地,该胶束还可进一步负载其他疏水性小分子化合物;
优选地,该胶束粒径为61.6±8.0nm;
优选地,该胶束在血清中电位为-6.1mV;
优选地,该胶束对半花菁类染料的包封率为61.4%,载药量为6.11%。
优选地,在PBS 7.4中胶束稳定性优异,一周之内粒径均稳定在50nm以下;而血清中胶束稳定性略差,在第5天粒径出现增大趋势,但总体仍在100nm以下。
优选地,该胶束可在还原剂维生素C存在条件下释放奥沙利铂,同时胶束结构被破坏。
优选地,DHAA-PEG-PLGA在所有胶束构建材料的掺杂比为20%。
本发明提供了一种可供临床或科研选择的基于奥沙利铂偶联性两亲聚合物的胶束,有益于解决目前临床和纳米医药工业出现奥沙利铂制剂化方面的问题。
本发明通过实施例表明:所述的基于奥沙利铂偶联性两亲聚合物的胶束,具有以下优点:
1)利用化学共价键将奥沙利铂固载在两亲聚合物材料中,在血液循环中保持更好的稳定性,在还原微环境中特异释放。
2)基于奥沙利铂偶联性两亲聚合物的胶束还可供负载其他类型的疏水性小分子,为奥沙利铂和其他抗肿瘤药物的联合应用打下基础。
3)本发明的基于PEG、PLGA胶束系统,作为FDA批准的临床可用的辅料,可实现体内长循环和拥有体内降解能力,生理毒性较低。
附图说明
图1,氧化型奥沙利铂前药的合成。
图2,合成异氰酸酯活化型PEG的反应式。
图3,PEG偶联奥沙利铂前药的合成。
图4,奥沙利铂偶联PEG和PLGA两亲性聚合物的合成。
图5,氧化型奥沙利铂前药HO-Pt-COOH的核磁图。
图6,氧化型奥沙利铂前药HO-Pt-COOH的质谱图。
图7,m-PEG-Pt-PLGA(左)、N3-PEG-Pt-PLGA(右上)、DHAA-PEG-Pt-PLGA(右下)。
图8,PLGA,PEG-Pt,PEG,PEG-Pt-PLGA的GPC结果。
图9,聚合物PEG-Pt-PLGA的还原响应性。
图10,芘荧光探针法测定CMC。
图11,聚合物胶束的透射电镜图。
图12,(a)空胶束及载药胶束分别在PBS 7.4和血清中的Zeta电位;(b)DYE-DNS的结构。
图13,胶束的EDS-Mapping元素分析。
图14,胶束稳定性考察。
图15,还原条件(Vc 5mM)下胶束粒径的变化。
图16,不同Vc浓度下胶束中奥沙利铂的释放曲线。
图17,流式细胞仪考察DHAA最佳修饰度。
具体实施方式
以下通过具体实施方式详细说明本发明的技术方案,应理解以下的具体实施方案仅为示例性,任何改动或变化只要不脱离本发明的技术方案设计,都应在本发明权利的要求保护范围之内。
实施例1
采用过氧化氢对奥沙利铂氧化,制得氧化型奥沙利铂前药HO-Pt-OH,再通过控制投料量使其中一个羟基与琥珀酸酐开环成酯,制得琥珀酸酯修饰的氧化型奥沙利铂前药HO-Pt-COOH。电子天平称取奥沙利铂100mg(0.25mmol),在黑暗环境下加入到4mL浓度为30%的过氧化氢水溶液中,室温搅拌反应24h。将整个混合体系冷冻干燥,得到淡黄色粉末状的HO-Pt-OH。称取所得HO-Pt-OH 135mg(0.31mmol),另称琥珀酸酐34mg(0.35mmol),一起加入到6mL无水DMF中,50℃油浴搅拌反应24h。冷至室温后经乙醚沉淀3次,得到白色粉末状的HO-Pt-COOH。利用1H NMR和质谱对所得产物进行结构鉴定。
实施例2
利用三光气与伯胺形成异氰酸酯的反应制备了N3-PEG-NCO及m-PEG-NCO。在氩气保护下将两口瓶中的无水甲苯加热至回流(110℃)。电子天平称取三光气38.2mg(0.13mmol)和N3-PEG-NH2 5k 530mg(1mmol)溶于无水甲苯中,得到澄清液体。将三光气的无水甲苯溶液加入两口瓶。然后用注射器将N3-PEG-NH25k的无水甲苯溶液在1h内匀速滴加至两口瓶。混合物在110℃回流下搅拌反应2h。待反应结束,全部转移至单口瓶并用旋转蒸发仪减压除去溶剂,用真空干燥箱进一步除去溶剂。得到的N3-PEG-NCO直接用于下一步反应。m-PEG-NCO同法合成,只需用m-PEG-NH2 5k替换N3-PEG-NH2 5k。
实施例3
利用异氰酸酯与羟基生成氨基甲酸酯的反应,将2.1中所得HO-Pt-COOH连接在PEG上得到N3-PEG-Pt-COOH及m-PEG-Pt-COOH。用适量无水DMF溶解2.2所得PEG-NCO,电子天平称取HO-Pt-COOH 451mg(0.85mmol)溶于无水DMF中得到澄清液体,二者混合于两口瓶中,再加入催化量的二月桂酸二丁基锡(DBTL)。在氩气保护下油浴50℃搅拌反应2h。旋转蒸发和真空干燥除去DMF,残余物在乙醚中沉淀3次,得到白色固体N3-PEG-Pt-COOH。m-PEG-Pt-COOH同法合成。
实施例4
通过羧基与羟基的酯化反应,将PLGA-OH连接至N3-PEG-Pt-COOH或m-PEG-Pt-COOH上,得到N3-PEG-Pt-PLGA及m-PEG-Pt-PLGA。电子天平称取N3-PEG-Pt-COOH 558mg(0.1mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)28.8mg(0.15mmol)、1-羟基苯并三唑(HoBt)20mg(0.15mmol)溶于无水DMF中,再加入三乙胺21μL(0.15mmol),在室温下搅拌2h。然后加入PLGA-OH 5k 500mg(0.1mmol),室温下搅拌反应过夜。混合物用截留分子量8k Da的蛇皮透析袋封装,于1L DMF中透析48h。透析袋内溶液转移至单口瓶,在真空干燥下浓缩至只有少量DMF,然后将其完全转入乙醚中沉淀3次,得到白色固体N3-PEG-Pt-PLGA。m-PEG-Pt-PLGA同法合成。
再通过炔基与叠氮的Click反应,将炔基修饰的DHAA与N3-PEG-Pt-PLGA连接得到DHAA-PEG-Pt-PLGA。
实施例5
将聚合物PEG-Pt-PLGA溶解在适量PBS 7.4中,一式三份,在氮气保护下,向其中两份分别加入计算量的维生素C(Vc),使Vc浓度为5mM,第三份作为对照,在37℃下,100rpm摇床上,Vc组分别放置12h和24h,对照组放置24h。直接冷冻干燥,用GPC测量分子量分布变化。
实施例6
临界胶束浓度(CMC)的测定:采用芘荧光探针法测定胶束的CMC。配制浓度为0.1mM(21.2mg/L)芘的丙酮溶液。配制浓度为0.5mg/mL的PEG-Pt-PLGA聚合物溶液。按表1的配比加入10mL EP管中混匀,再加去离子水至总体积为10mL。遮光在37℃摇床孵育过夜,于荧光分光光度计检测334nm激发波长下,在373nm和384nm处的荧光强度,二者做比得到I1/I3作为纵坐标,以聚合物浓度(0.25,0.5,1,2.5,5,10,25,50,100,250μg/mL)为横坐标,绘制趋势图,计算得到CMC。
由于芘不溶于水,当两亲性聚合物浓度达到临界胶束浓度时,会骤然增大芘的溶解度,从而改变其荧光强度,因此,通过以I1/I3为纵坐标、聚合物浓度为横坐标绘制散点图,如图11所示,对低浓度区和高浓度区分别进行线性拟合,所得两条趋势线的交界点,即是胶束的CMC。计算得CMC为11.8μg/mL。
实施例7制备聚合物胶束
将聚合物材料PEG-Pt-PLGA完全溶解在甲醇中,在旋转蒸发仪上减压除去甲醇,使聚合物均匀地覆于圆底瓶内壁形成薄膜,置真空干燥箱内尽量除尽甲醇;加入适量去离子水,使聚合物浓度高于CMC的量,超声处理1h,再经0.22μm水系微孔滤膜过滤,得到分散均匀、具有乳光的空胶束溶液;将疏水性探针DYE-DNS与聚合物材料PEG-Pt-PLGA一起完全溶解在甲醇中,后续同法,注意避光操作,制备得到包有荧光探针DYE-DNS的载药胶束溶液。
实施例8
由于奥沙利铂直接共价连接在聚合物内,因此可通过计算奥沙利铂在聚合物中的分子量占比,估算聚合物胶束中奥沙利铂的载药量。使用HPLC法测定聚合物胶束对荧光探针DYE-DNS的包封率和载药量。HPLC条件:流动相为0.1%三氟乙酸水溶液/乙腈(ACN),梯度洗脱(洗脱时流动相中ACN比率随时间变化曲线如图10所示);流速1mL/min;紫外检测器250nm;进样量20μL;柱温25℃;检测时间20min;DYE-DNS的保留时间为min。
以乙腈为溶剂,分别配制浓度为0.16、0.4、0.8、2、4、10、20μg/mL的DYE-DNS标准品溶液,绘制其荧光强度-浓度标准曲线,用于计算。按4.2的方法制备载药胶束的PBS溶液,取一定量,冷冻干燥称重得胶束总量,即w(胶束中的药)+w(聚合物)另取一定量经5000rpm离心5min后,取上清液20μL进样HPLC测定计算未包入药量。将过量Vc加入到一定量载药胶束的PBS 7.4溶液中,在氩气保护下,37℃,100rpm摇床上避光孵育12h,取20μL进样HPLC测定峰面积,计算总药量,总药量-未包入药量=胶束中药量。
根据结果计算:包封率=w(胶束中的药)/w(初始所加药);载药量=w(胶束中的药)/w(胶束中的药)+w(聚合物)。
实施例9
通过粒径变化考察胶束还原响应性:将制得的并经过0.22μm滤膜过滤的胶束溶液稀释至PBS 7.4中,在氮气保护下加入维生素C使浓度达到5mM,在37℃、100rpm摇床上孵育24h后,通过DLS测量粒径分布,考察粒径变化,以不加Vc孵育作为对照。
定量奥沙利铂的HPLC条件:流动相为甲醇:水=10:90,流速1mL/min,紫外检测器250nm,测定10min,奥沙利铂保留时间为4min。配制浓度为100μg/mL的奥沙利铂标准品溶液,对半稀释8次得到浓度分别为50、25、12.5、6.25、3.125、1.56、0.78、0.39μg/mL的奥沙利铂标准品溶液,经HPLC测定并绘制标准曲线,用于后续奥沙利铂的定量。
将制得的并经过0.22μm滤膜过滤的胶束溶液稀释至PBS 7.4中,在氮气保护下加入不同量Vc使浓度分别为0、0.5、1、5mM,在37℃、100rpm摇床上孵育,并在第0、1、2、4、6、8、10、12、16、24、36h分别取样20μL用于HPLC测定奥沙利铂的浓度,n=3,绘制不同Vc浓度下奥沙利铂的释放曲线。
HCT-116细胞的培养:HCT-116细胞悬浮于含有10%FBS、1%双抗的DMEM培养基中,以2万个细胞/孔的密度接种于24孔板中,于CO2 5%、37℃培养,至融合度达70%时可用于实验。
实施例10
通过流式考察DHAA最佳修饰度:将含DHAA靶头的聚合物DHAA-PEG-Pt-PLGA与无靶头聚合物m-PEG-Pt-PLGA以不同DHAA的占比(0%、10%、20%、50%、100%)混合,并与荧光探针DYE-DNS共溶于甲醇得到澄清液体,以薄膜水化法制得不同DHAA修饰度的载药胶束。在实验前12h将HCT-116细胞的培养基换为无糖DMEM拉平细胞状态,然后加入不同DHAA修饰度的胶束孵育30min后,用无糖Hank’s洗去胶束。再用胰酶消化细胞,无糖Hank’s稀释终止消化,3000rpm离心10min后弃上清,用无糖Hank’s重悬细胞。使用流式细胞仪对结果定量,激发波长650nm,每次分析收集1万个细胞,未处理的细胞作为空白对照。
经流式计数结果显示,与0%修饰度相比,DHAA基团的修饰增加了癌细胞HCT-116对胶束的摄取,这是由细胞表面高表达的GLUT1转运体介导的;而当修饰比例高于20%时,各组的细胞摄取之间无明显差异,因此,结果表明,20%的DHAA修饰度可增加癌细胞对胶束的摄取,赋予胶束对癌细胞的主动靶向能力。
Claims (9)
1.一种基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于:该胶束的构筑基元为两亲性的聚合物材料,所述的两亲性的聚合物材料是亲水性聚乙二醇(PEG)和疏水性聚乳酸-羟基乙酸共聚物(PLGA)偶联体,其中,PEG的平均分子量为5000, PLGA的平均分子量为4500,
奥沙利铂偶联性两亲聚合物的制备方法为:
(1)采用过氧化氢对奥沙利铂氧化,制得氧化型奥沙利铂前药HO-Pt-OH,再通过控制投料量使其中一个羟基与琥珀酸酐开环成酯,制得琥珀酸酯修饰的氧化型奥沙利铂前药HO-Pt-COOH;
(2)利用三光气与伯胺形成异氰酸酯的反应制备m-PEG-NCO;
(3)利用异氰酸酯与羟基生成氨基甲酸酯的反应,将所得HO-Pt-COOH连接在PEG上得到m-PEG-Pt-COOH;
(4)通过羧基与羟基的酯化反应,将PLGA-OH连接至m-PEG-Pt-COOH上,得到奥沙利铂偶联性两亲聚合物m-PEG-Pt-PLGA。
2.根据权利要求1所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的PEG和PLGA两亲性聚合物偶联体中,该两亲性聚合物的临界胶束浓度为11.8μg/mL。
3.根据权利要求1所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的PEG-Pt-PLGA偶联体还修饰维生素C类似物DHAA,其中DHAA-PEG-Pt-PLGA在胶束构建材料的掺杂比为20%。
4.根据权利要求1所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的胶束还负载其他疏水性小分子化合物。
5.根据权利要求1所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的胶束粒径为61.6±8.0nm。
6.根据权利要求1所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的胶束在血清中电位为-6.1mV。
7.根据权利要求1所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的胶束还对亲水性的抗肿瘤化疗药物奥沙利铂有效负载,并优化其体内分布过程。
8.根据权利要求4所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的胶束对半花菁类染料的包封率为61.4%,载药量为6.11%。
9.根据权利要求6所述的基于奥沙利铂偶联性两亲聚合物的胶束,其特征在于,所述的胶束在还原剂维生素C存在条件下释放奥沙利铂,同时胶束结构被破坏。
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