CN114456373A - 一种基于亮氨酸的聚酯酰胺的纳米递药体系及其制备方法和应用 - Google Patents
一种基于亮氨酸的聚酯酰胺的纳米递药体系及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于亮氨酸的聚酯酰胺的纳米递药体系及其制备方法和应用。本发明提供了一种基于亮氨酸的聚酯酰胺的制备方法:合成二元酸二硝基苯酯(单体Ⅰ);合成二亮氨酸二甲基苯磺酸盐酯(单体Ⅱ);将单体Ⅰ和单体Ⅱ按一定比例投入到反应体系中,以三乙胺为催化剂,在70~80℃搅拌反应约24~48h,得到基于亮氨酸的聚酯酰胺高分子。这种聚合物结构可控,可生物降解,具有良好的生物相容性,能够有效装载药物,得到的纳米粒载药量高,药物释放性质好,具备高效肿瘤靶向和高效抑制肿瘤细胞生长的特点,降低了化疗药物对正常组织的毒性,绿色安全。
Description
技术领域
本发明属于生物医用材料技术领域,具体涉及一种适用于高效装载抗肿瘤类药物的基于亮氨酸的聚酯酰胺纳米递药体系及其制备方法和应用。
背景技术
近年来,恶性肿瘤的发病率呈现上升的趋势,已经成为了目前全世界的主要死亡原因之一,是严重危害人类生命健康、制约社会经济发展的一大类疾病。目前恶性肿瘤的治疗方式有:手术、化疗和放疗,其中化疗药物是很常用的治疗方法。在使用化疗药物时,最重要的是提高其装载效率以及降低其在非作用部位的毒副作用。因此,寻找合适的药物载体是应用是关键。
阿霉素(Dox)是一种广泛应用的抗肿瘤药物,它是一种抗肿瘤抗生素,可抑制RNA和DNA的合成,对RNA的抑制作用最强,抗瘤谱较广,对多种肿瘤均有作用,属周期非特异性药物,对各种生长周期的肿瘤细胞都有杀灭作用。主要适用于急性白血病,对急性淋巴细胞白血病及粒细胞白血病均有效,对恶性淋巴瘤乳腺癌、肉瘤、肺癌、膀胱癌等其他各种癌症也都有一定疗效。但是,阿霉素作为广谱抗肿瘤药,对机体可产生广泛的生物化学效应,具有强烈的细胞毒性作用,可引起机体多种不良反应,如白细胞和血小板减少、毛发脱落、心律失常、恶心、食欲减退等。因此,寻找一种递送方法,使得维持阿霉素良好疗效的同时降低其毒副作用,是非常重要的。
传统的药物运输方式存在一定的缺陷:是药物循环时间过短导致其在肿瘤组织中富集浓度不足,且一般抗肿瘤药物都有一定细胞毒性,对体内正常组织细胞产生毒副作用。目前纳米药物的热点主要是药物制剂、治疗制剂和诊断制剂的理论传输途径和靶向功能的研究。理想的抗肿瘤纳米材料进入机体后,能随着其所到达组织部位的不同有效地自我调控其与生物系统的相互作用;在血液循环过程中,纳米药物应尽可能避免或减少和巨噬细胞等吞噬细胞相互作用;而当其到达肿瘤组织时,纳米药物应能够增强与肿瘤细胞的相互作用;在肿瘤细胞内,纳米药物应能够快速释放出活性药物分子,以增强与药物靶标的相互作用。尽管这种设计原则获得研究人员的一致认可,但如何实现这一原则的纳米药物载体仍极具挑战。
聚酯酰胺是一种同时具有酯键和酰胺键的高分子聚合物,酯键的存在使其容易水解,因此可生物降解,生物相容性好。酰胺键则使其拥有较好的机械性能和热力学性能,是良好的药物载体。
发明内容
本发明要解决的技术问题是针对现存的抗肿瘤类药物递送效率低、毒副作用大等问题,以阿霉素为代表药物,设计一种可生物降解的聚合物分子,实现抗肿瘤类药物的高效装载以及理想释放,最大程度上降低毒副作用。
本发明的第一个目的是提供一种基于亮氨酸的聚酯酰胺聚合物。
本发明的第二个目的是提供上述基于亮氨酸的聚酯酰胺聚合物的制备方法。
本发明的第三个目的在于提供上述聚酯酰胺聚合物在制备或作为药物载体中的应用。
本发明的第四个目的是提供一种基于上述聚酯酰胺聚合物的纳米载体。
本发明的第五个目的是提供一种基于上述聚酯酰胺聚合物的载药纳米体系。
本发明上述目的通过以下技术方案实现:
一种基于亮氨酸的聚酯酰胺聚合物,其化学结构式如式1所示:
式中,n、x、y均表示聚合度,其中,n的取值范围为1~75,优选为30~75,x与y的取值范围均为1~12,优选为2~10;*表示连接位点。
上述基于亮氨酸的聚酯酰胺聚合物的制备方法,包括以下步骤:
S1.合成二元羧酸二对硝基苯基酯单体(Nx);
S2.合成二亮氨酸二对甲苯磺酸盐酯单体(Leu-y);
S3.将二元羧酸二对硝基苯基酯单体和二亮氨酸二对甲苯磺酸盐酯单体加入反应溶剂中,65~85℃搅拌条件下逐滴加入三乙胺作为反应催化剂,在60~80℃下加热反应24~96h,即获得所述的基于亮氨酸的聚酯酰胺聚合物(x-Leu-y-PEA)。
实验发现,本发明二元羧酸二对硝基苯基酯单体和二亮氨酸二对甲苯磺酸盐酯单体的产率分别达95%、72%以上,且纯度较好,制备出的聚酯酰胺聚合物适用于制备纳米粒溶液。若反应时间短或反应温度低可能导致单体反应不充分而产生副产物,降低聚合物产率和纯度。
在本发明较佳的实施例中,步骤S1所述二元羧酸二对硝基苯基酯单体的制备方法为:将2~3倍于二元酰氯当量的对硝基苯酚与2~3倍于二元酰氯当量的三乙胺加入-85~-70℃的冰冻丙酮中,得到混合液A;将二元酰氯加入-85~-70℃的冰冻丙酮中,混合均匀;然后逐滴加入所述混合液A;-85~-70℃搅拌反应1~5h后转至20~30℃搅拌10~16h。如果反应时间不足,反应物不能充分反应,产率较低,且得到的产物易含有较多杂质和副产物。
在本发明较佳的实施例中,步骤S1所述二元酰氯选自乙二酰氯、丁二酰氯或辛二酰氯,反应完成后分别得到乙二酸二对硝基苯基酯(N2),丁二酸二对硝基苯基酯(N4)和辛二酸二对硝基苯基酯(N8)。
在本发明较佳的实施例中,步骤S1得到二元羧酸二对硝基苯基酯单体后将其洗涤,50~60℃真空干燥至恒重,并利用乙腈重结晶进行纯化。
在本发明较佳的实施例中,步骤S1所述反应的条件为-78℃搅拌反应2h后转至25℃搅拌10~16h。
在本发明较佳的实施例中,步骤S2所述二亮氨酸二对甲苯磺酸盐酯单体的制备方法为:以对甲苯磺酸一水合物作为催化剂和氨基保护剂,将L-亮氨酸、1/2~1/3L-亮氨酸当量的二元醇、2~4倍于L-亮氨酸当量的甲苯磺酸一水合物在甲苯中于110~150℃反应16~72h。如果反应时间不足,反应物不能充分反应,产率较低,且得到的产物易含有较多杂质和副产物。
在本发明较佳的实施例中,步骤S2所述二元醇选自乙二醇、1,4-丁二醇、1,6-己二醇中的一种或几种,反应完成后分别得到二亮氨酸二对甲苯磺酸盐乙酯(Leu-2)、二亮氨酸二对甲苯磺酸盐丁酯(Leu-4)以及二亮氨酸二对甲苯磺酸盐己酯(Leu-6)。
在本发明较佳的实施例中,步骤S2所述反应温度为120~140℃,反应时间为24~30h。
在本发明较佳的实施例中,步骤S2得到二亮氨酸二对甲苯磺酸盐酯单体后,将其在70~80℃(优选75℃磁力搅拌)加热搅拌条件下溶解于沸水中,并在2~8℃条件下析出沉淀,重复2~4次,再于55~65℃真空干燥24~48h,干燥条件优选为60℃真空干燥24~36h。
在本发明较佳的实施例中,步骤S3所述二元羧酸二对硝基苯基酯单体和二亮氨酸二对甲苯磺酸盐酯单体的质量比为1:2~2:1,优选为1:1。
在本发明较佳的实施例中,步骤S3所述反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺(DMAC)或二甲基亚砜(DMSO)中的一种或几种。
在本发明较佳的实施例中,步骤S3得到聚酯酰胺聚合物后,将其倒入预冷的乙酸乙酯中析出沉淀,弃上清,将产物用甲醇复溶,复提纯除杂3~4次后,将产物于50~60℃真空干燥24~48h,干燥条件优选为60℃真空干燥24~36h。真空干燥温度和时间可随具体情况调控。
上述聚酯酰胺聚合物在制备或作为药物载体中的应用。
在本发明较佳的实施例中,所述的载体为纳米载体。
一种基于上述聚酯酰胺聚合物的纳米载体的制备方法,通过如下任一种方式实现:
(I)将所述聚酯酰胺聚合物溶于有机溶剂中,得到聚酯酰胺聚合物溶液;然后在搅拌条件下,将聚酯酰胺聚合物溶液滴加至含有稳定剂的水溶液中,使其自组装成纳米粒,得到聚酯酰胺聚合物药物输送载体;
或
(II)将所述聚酯酰胺聚合物和稳定剂分别溶解于有机溶剂中,得到聚酯酰胺聚合物溶液和稳定剂溶液;然后将两者混合均匀后滴加至水中,使其自组装成纳米粒,得到聚酯酰胺药物输送载体。
一种纳米载体,通过上述方法制备得到。
一种基于上述聚酯酰胺聚合物的载药纳米体系的制备方法,通过如下任一种方式实现:
(I)将所述聚酯酰胺聚合物、抗肿瘤药物和稳定剂分别溶解到有机溶剂中,得到聚酯酰胺聚合物溶液、抗肿瘤药物溶液和稳定剂溶液;然后将三种溶液混合均匀后滴加至水中,使其自组装成载药纳米粒,得到聚酯酰胺载药纳米体系;
或
(II)将上述聚酯酰胺聚合物和抗肿瘤药物分别溶解到有机溶剂中,得到聚酯酰胺聚合物溶液和抗肿瘤药物溶液;然后将聚酯酰胺聚合物溶液和抗肿瘤药物溶液滴加到含有稳定剂的水溶液中,使其自组装成纳米粒,得到聚酯酰胺载药纳米体系。
一种载药纳米体系,通过上述方法制备得到。
对于上述纳米载体和载药纳米体系的制备方法,在本发明较佳的实施例中,方式(I)和(II)中所述的稳定剂为聚乙烯醇(PVA)、两性离子活性剂或DSPE-PEG(二硬脂酰基磷脂酰乙醇胺-聚乙二醇);优选为DSPE-PEG2000。
在本发明较佳的实施例中,所述的两性离子活性剂优选为羧基甜菜碱或磺基甜菜碱。
在本发明较佳的实施例中,方式(I)和(II)中所述的稳定剂的用量为占聚酯酰胺聚合物质量的0~75%(不包括0);优选为占亮氨酸聚酯酰胺聚合物质量的25~50%;更优选为占氨基酸聚合物质量的50%。
在本发明较佳的实施例中,方式(I)和(II)中所述的有机溶剂为二甲基亚砜(DMSO)、N,N-二甲基甲酰胺(DMF)和四氢呋喃(THF)中的一种或多种;优选为二甲基亚砜(DMSO)。
在本发明较佳的实施例中,方式(I)和(II)中所述的聚酯酰胺聚合物溶液的浓度为5~50mg/mL;优选为10~50mg/mL;更优选为10~20mg/mL。
在本发明较佳的实施例中,方式(I)和(II)中所述的稳定剂溶液的浓度为5~50mg/mL;优选为10~50mg/mL;更优选为10~20mg/mL。
在本发明较佳的实施例中,所述的制备方法还包括将得到的产物去除溶剂的步骤,具体为:将自组装成的纳米粒置于超滤管中,离心,重复3次以上,使有机溶剂的含量在千分之一以下。
在本发明较佳的实施例中,所述是超滤管为截留分子量为100kDa的超滤管。
在本发明较佳的实施例中,所述的离心的条件为:2000~3000rpm离心8~10min;优选为:2500rpm离心10min。
其中,对于所述的载药纳米体系的制备方法:
在本发明较佳的实施例中,方式(I)和(II)中所述的抗肿瘤药物溶液的浓度为5~50mg/mL;优选为10~50mg/mL;更优选为10~20mg/mL。
在本发明较佳的实施例中,方式(I)和(II)中所述的聚酯酰胺聚合物与抗肿瘤药物的质量比为9:1~2:1;优选为5:1。聚酯酰胺聚合物和抗肿瘤药物的质量比过大,则载药量较低,所成纳米粒粒子数较少,过小则包封率较小,造成药物的浪费,并且所得纳米粒不稳定,易聚沉。
在本发明较佳的实施例中,对所载的抗肿瘤药物没有特殊限制,所述的药物包括亲水性药物和疏水性药物;所述亲水性药物包括但不限于盐酸阿霉素、盐酸吉西他滨、盐酸伊立替康、氟尿嘧啶或香菇多糖等药物;所述疏水性药物包括但不限于紫杉醇(PTX)、多西紫杉醇、甲氨蝶呤、喜树碱、阿霉素、姜黄素等药物。
在本发明中,以亮氨酸聚酯酰胺聚合物为药物载体,负载典型抗肿瘤药物阿霉素(Dox),利用纳米沉淀法,以DSPE-PEG和聚合物亲水性部分为亲水性外壳,以疏水性药物为内核,在水中自组装成具有核壳结构。所述的载药纳米粒的粒径为90~110nm,拥有较高的比表面积,载药量高,稳定性良好,生物相容性好,是优良的载药体系,不仅实现了疏水药物的可控载药,改善了药物的溶解性,极大的提高了疏水药物的可利用性,还可以大大提高纳米粒在血液中的循环时间,从而提高肿瘤部位的药物聚积,提高治疗效果。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明基于一种天然氨基酸亮氨酸设计了一类聚酯酰胺高分子,并调整合成所用的二酸以及二醇的结构构建了一个高分子载体库。该聚合物具有较好的生物可降解性、生物相容性以及结构可调控性。
(2)本发明利用基于亮氨酸的聚酯酰胺聚合物负载抗肿瘤药物,制备而成的纳米粒载体具有载药量高且尺寸稳定,能够通过肿瘤组织的增强渗透滞留效应(EPR)较好地聚集在肿瘤部位,在提高化疗药物的生物利用度且降低化疗药物对正常组织的毒性的同时增强了纳米药物载体对肿瘤细胞生长抑制的特点。
(3)本发明利用基于亮氨酸的聚酯酰胺聚合物负载抗肿瘤药物,可以通过激活mTOR通路,抑制药物所诱导的细胞自噬,从而提高治疗过程对药物的敏感性。
(4)本发明同时改善了药物的溶解性,极大的提高了疏水药物的可利用性,还可以大大提高纳米粒在血液中的循环时间,从而提高肿瘤部位的药物聚积。另外,肿瘤组织具有不同于正常组织的微环境,与正常细胞相比,肿瘤组织呈弱酸性,酰胺键在肿瘤细胞质中较低pH下会加快药物释放,从而引发药物的释放至肿瘤细胞,并且随后可高效诱导肿瘤细胞凋亡。
(5)本发明步骤简单,易于操作,重现性好,作为优秀的药物递送系统,在肿瘤治疗等生物医学应用中有着良好的前景。
附图说明
图1为不同二元羧酸二对硝基苯基酯单体、不同二亮氨酸二对甲苯磺酸盐酯单体和基于亮氨酸聚酯酰胺聚合物的合成步骤。
图2为一种基于亮氨酸的聚酯酰胺聚合物(8L6)的1H-NMR图谱。
图3为制备的基于亮氨酸的聚酯酰胺的纳米载体(8L6 NPs)粒经分布及透射电镜图。
图4为制备的负载Dox的基于亮氨酸的聚酯酰胺的纳米载体(Dox@8L6 NPs)的粒经分布及透射电镜图。
图5为不同条件下制备的Dox@8L6 NPs的稳定性试验结果图。
图6为制备的Dox@8L6 NPs在不同pH条件下的释放结果。
图7为制备的Dox@8L6 NPs对THP-1细胞(急性单核细胞白血病细胞)的毒性实验结果。
图8为THP-1细胞对制备的载药纳米粒Dox@8L6 NPs的激光共聚焦显微镜下的摄取情况图。
图9为THP-1细胞对制备的载药纳米粒Dox@8L6NPs的流式细胞仪的定量摄取情况。
图10为制备得到的载药纳米粒Dox@8L6NPs作用于THP-1细胞的凋亡结果图。
图11为白血病干细胞(LSC)的分类以及检测的mTOR通路和自噬相关蛋白的表达结果
图12为Dox@8L6 NPs对急性骨髓性白血病(AML)小鼠的体内治疗效果图;其中,图A是AML小鼠的给药方案,图B是不同时间点AML小鼠外周血液中GFP+白血病细胞的比例,图C是外周血液涂片结果,图D是不同时间点AML小鼠的体重变化情况,图E是第21天时AML小鼠骨髓中GFP+白血病细胞的比例。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明,本发明所用试剂和原材料均可通过市售获得。
实施例1基于亮氨酸的聚酯酰胺聚合物的制备
1、基于亮氨酸的聚酯酰胺聚合物的制备方法,包括以下步骤:
(1)二元羧酸二对硝基苯基酯单体(Nx)的合成:
(a)称取一定量的乙二酰氯(或丁二酰氯或辛二酰氯);量取略大于2倍乙二酰氯当量的对硝基苯酚和略大于2倍乙二酰氯当量的三乙胺加入-85~-70℃的冰冻丙酮中,室温下搅拌均匀,然后用干冰将其保存在-78℃下,得到混合液A;
(b)将前面称取的二元酰氯加入到-85~-70℃的冰冻丙酮溶液中,混合均匀,然后逐滴加入所述混合液A,在-78℃搅拌下搅拌2h后转至25℃搅拌过夜(10~16h)。
(c)将反应后的溶液加入装有足量蒸馏水的大烧杯中,产物沉淀后过滤,洗涤,然后置于50℃的真空干燥箱中烘干;
(d)利用乙腈将产物重结晶3次进行纯化,分别得到乙二酸二对硝基苯基酯(N2),丁二酸二对硝基苯基酯(N4)和辛二酸二对硝基苯基酯(N8),产率分别为95%、98%、99%。
(2)二亮氨酸二对甲苯磺酸盐酯单体(Leu-y)的合成:
(a)称取适量的L-亮氨酸,并量取二分之一亮氨酸当量的乙二醇(或称取丁二醇或己二醇),再称2~3倍亮氨酸当量的对甲苯磺酸一水合物,均加入三口烧瓶;
(b)将足量的甲苯加入三口烧瓶中,安装好冷凝装置,加热到至130℃,均匀搅拌24h;
(c)反应结束后,冷却至室温,倒出甲苯,得到白色固体;
(d)在75℃、搅拌条件下将产物溶解于沸水中,随后置于4℃下沉淀,重复3次,最后将产物置于60℃真空干燥箱中烘干24h,分别得到二亮氨酸二对甲苯磺酸盐乙酯(Leu-2)、二亮氨酸二对甲苯磺酸盐丁酯(Leu-4)以及二亮氨酸二对甲苯磺酸盐己酯(Leu-6),产率分别为72%,80%,86%。
(3)基于亮氨酸的聚酯酰胺聚合物的制备:
(a)根据质量比1:1分别称取一定量的上述制备得到的二元羧酸二对硝基苯基酯单体和二亮氨酸二对甲苯磺酸盐酯单体,混合均匀放入20mL玻璃反应瓶,加入足量的反应溶剂DMF(N,N-二甲基甲酰胺),搅拌,加热至70℃,使单体完全溶解呈透明溶液;制备原料中的反应溶剂DMF可以用其它有机溶剂,如可用N,N-二甲基乙酰胺或二甲基亚砜所代替;
(b)在快速搅拌下向溶液中逐滴加入适量三乙胺;
(c)待搅拌均匀,溶液颜色变黄后,停止搅拌,将反应体系置于70℃下反应24h;
(d)将反应后的溶液倒入预冷的乙酸乙酯中使产物沉淀,弃上清,将产物重新溶解于甲醇中,再用冷的乙酸乙酯使产物沉淀,重复提纯2次,最后将产物置于60℃真空干燥箱中烘干24h,可得到一系列基于亮氨酸的聚酯酰胺聚合物。
2、结果
步骤(1)、(2)、(3)的合成步骤如图1所示;其所合成的其中一种基于亮氨酸的聚酯酰胺聚合物(8L6)的1H-NMR图谱如附图2所示,说明本发明中基于亮氨酸的聚酯酰胺聚合物的成功合成。
实施例2基于亮氨酸的聚酯酰胺的纳米载体的制备
1、基于亮氨酸的聚酯酰胺的纳米载体的制备过程包括以下步骤:
(1)将实施例1制备得到的一种亮氨酸聚酯酰胺(8L6)和表面稳定剂DSPE-PEG2000分别溶于二甲基亚砜(DMSO)中,分别配成10mg/mL的溶液备用;
(2)将上述配制的亮氨酸聚酯酰胺(8L6)与表面稳定剂DSPE-PEG2000溶液按照一定比例混合后在1000rpm转速下缓慢滴加至水溶液中,该复合物在水溶液中通过纳米沉淀法自组装形成纳米体系;其中,DSPE-PEG2000的质量为8L6质量的50%;
(3)将所得纳米粒溶液置于截留分子量(MWCO=100kDa)的超滤管中,2500rpm超滤3次,每次10min,使DMSO含量在1‰以下,最后得到8L6 NPs载体。
2、纳米粒表征结果
利用纳米粒经仪和透射电镜对其物理性质进行表征。
3、结果
利用纳米粒经仪和透射电镜对8L6NPs的粒径和形态进行表征,结果如图3所示,所制得的纳米粒子粒径约为112nm,呈类圆形,大小相对均一。
实施例3基于亮氨酸的聚酯酰胺的载药纳米体系的制备
1、基于亮氨酸的聚酯酰胺的载药纳米体系的制备过程包括以下步骤:
(1)本实施例以抗肿瘤药物阿霉素(Dox)为代表进行制备,将Dox、表面稳定剂DSPE-PEG2000,实施例1制备得到的亮氨酸聚酯酰胺聚合物(8L6)分别溶于DMSO中,配成10mg/mL的溶液备用;
(2)将等浓度的三种溶液(8L6,Dox,DSPE-PEG2000),按照聚合物8L6和抗肿瘤药物Dox的质量比为9:1,5:1,3:1,2:1不同比例混合后,按DSPE-PEG2000占聚合物8L6质量的0~75%进行混合,在1000rpm转速下缓慢滴加至水溶液中,该复合物在水溶液中通过纳米沉淀法自组装形成纳米体系;其中,本案例较佳DSPE-PEG2000质量为8L6质量的50%;
(3)将所得纳米粒溶液置于截留分子量(MWCO=100kDa)的超滤管中,2500rpm超滤3次,每次10min,以除去未包封的Dox并使DMSO含量在千分之一以下,最后得到Dox@8L6NPs载药纳米粒。
2、载药纳米粒粒径载药测试、稳定性试验以及释药实验
(1)载药纳米粒粒径载药测试,测量不同质量比投料的载药纳米粒的粒径、PDI以及载药量。
(2)稳定性试验:将纳米粒置于PBS以及PBS+10%FBS的环境中,测量其七天内的粒径和PDI情况。
(3)释药实验:将Dox@8L6 NPs密封于透析袋中(MWCO:3500Da)。然后将透析袋分别置于pH值为5.0、6.8、7.4的不同环境中,这些环境由不同体积比的柠檬酸和磷酸氢钠溶液组成。将系统置于恒温振动筛(37℃,100rpm)中。然后,在特定时间点(0.25、0.5、1、2、4、6、8、12、24、48、96、120h),从缓冲液中取出500μl释放液,加入等量缓冲液,测定其中的Dox浓度。计算各时间点Dox的累积释放量。
3、结果
(1)在聚合物8L6和其他条件相同的条件下,以聚合物8L6与Dox的质量比为单变量,考察发现随着聚合物材料和Dox质量比对纳米粒稳定性、载药量有显著影响,当Dox含量逐渐增加时,载药量先是增加后逐渐降低,综合考虑载药量和包封率的问题,聚合物和Dox质量比为5:1时较好(表1)。利用纳米粒经仪和透射电镜对Dox@8L6 NPs的粒径和形态进行表征,结果如图4所示,所制得的纳米粒子粒径约为104nm,呈类圆形,大小相对均一。
表1载药量统计
(2)载药纳米粒的七天稳定性结果如图5所示,纳米粒在PBS中较稳定,在含FBS的PBS中粒径和PDI稍有增加,但变化量较小,证明载药纳米粒能够在生理条件下保持长时间的稳定。
(3)Dox@8L6 NPs在不同pH值下体外的释放情况如图6所示:Dox@8L6 NPs中的药物在pH 5.0时的释放效率显著高于pH 7.4。当pH值为5.0时,Dox@8L6 NPs快速释放Dox,在20h左右达到40%的释放效率,之后缓慢释放,最终稳定在60%左右的释放效率,并可长期保持,证明Dox@8L6 NPs在肿瘤细胞和微环境中能够快速释放Dox,并维持其较长时间。
实施例4载药纳米粒Dox@8L6 NPs的体外抗肿瘤效果评价
1、对实施例3制备得到的纳米载药体系(聚合物8L6和PTX质量比为5:1,DSPE-PEG2000质量为聚合物8L6质量的50%)对急性单核细胞白血病细胞(THP-1)进行细胞作用评价,具体步骤如下:
(1)细胞毒性实验:将状态良好的THP-1细胞接种在96孔板中(每孔5000个细胞),孵育过夜。并在每孔中加入不同浓度的Dox和相应浓度的Dox@8L6 NPs。37℃,5%CO2孵育48小时,每孔加入10μLCCK-8溶液,继续孵育4小时。使用多功能酶标仪测量每孔在450nm处的吸光度。
将THP-1细胞接种于15mm玻璃底细胞培养皿中,37℃培养24h,然后用DOX-HCl,DOX@8L6 NPs处理1h,2h,4h,8h,收集细胞,使用DAPI(1306,Thermo Scientific)染色,用4%8L6 NPs固定15分钟,通过高速共焦成像系统(Dragonfly CR-DFLY-202 2540)观察。
(2)细胞摄取(激光共聚焦显微镜):将密度为2×105的THP-1细胞加入共聚焦激光培养皿中孵育24h,然后分别加入Dox或Dox@8L6 NPs孵育4h。用磷酸盐缓冲液洗涤细胞后,加入DAPI孵育10min,然后使用激光共聚焦扫描显微镜(CLSM)观察药物的细胞摄取情况。
(3)细胞摄取(流式细胞仪):将密度为2×105的THP-1细胞加入孔板中孵育24h,然后分别加入Dox或Dox@8L6 NPs孵育1,2,4和8h,用磷酸盐缓冲液洗涤细胞后转移到流式管中,在流式细胞仪上检测细胞内Dox的荧光强度。
(4)细胞凋亡:根据Annexin V-FITC双染细胞凋亡检测试剂盒的对细胞进行染色并用流式细胞分析仪检测来检验细胞凋亡。具体为:将2mL密度为每孔5×105个细胞的THP-1细胞接种在6孔板中,在37℃、5%CO2的培养箱中培养24h后,将细胞用新鲜培养基(作为空白对照),Dox,8L6 NPs,Dox@8L6 NPs处理24h;然后消化细胞并重悬于缓冲液中,向细胞悬液中加入5μL AnnexinV-FITC,避光条件下温育15min后;向混合物中加入5μL碘化丙啶(PI);最后,使用流式细胞仪进行分析,每个组测定三次。
2、结果
(1)细胞毒性实验结果如图7所示:在指定药物治疗后不同时间点检测THP-1细胞的增殖情况。我们发现Dox能显著抑制THP-1细胞的增殖,同时,Dox@8L6 NPs对细胞增殖的抑制作用比纯药Dox更明显,对肿瘤细胞有较好的杀伤作用。
(2)激光共聚焦拍摄细胞摄取的结果如图8所示:Dox@8L6 NPs在4小时的细胞摄取高于其自由药物对应物,证明纳米粒能够被细胞较好的摄取,这为其发挥作用奠定基础。
(3)流式细胞仪的细胞摄取结果(图9)同样表明纳米粒能够被细胞更好地摄取,且在在治疗后8小时最明显(增加1.91倍)。
(4)图10为载药纳米粒的Dox@8L6诱导THP-1细胞凋亡的图,与游离Dox相比,载药纳米粒Dox@8L6诱导THP-1细胞调亡的能力更强。
实施例5载药纳米粒Dox@8L6 NPs抑制LSC自噬的验证以及体内抗肿瘤效果评价
1、LSC细胞中mTOR通路和自噬相关蛋白的表达结果检测
(1)白血病细胞移植建模后第14天,用Dox@8L6 NPs连续治疗AML小鼠3天,以PBS或DOX或8L6 NPs作为对照,第19天从骨髓中提取LSC并分类。AML小鼠模型的建立和LSC细胞的分离与提取方法参考文献“Forte,D.et al.Bone Marrow Mesenchymal Stem CellsSupport Acute Myeloid Leukemia Bioenergetics and Enhance Antioxidant Defenseand Escape from Chemotherapy.Cell metabolism 32,829-843.e829(2020)”。
(2)提取的LSC细胞用PBS洗涤并用RIPA裂解,用30μL蛋白G珠预先清除全细胞裂解液,提取蛋白并进行纯化。将等量的各组蛋白提取物用10%SDS-PAGE分离并转移到PVDF膜上。用5%脱脂牛奶在Tris缓冲盐水中与吐温20(TBST,pH 7.6)在室温下封闭膜1小时后与一抗(anti-LC3B I/II、anti-phospho-mTOR(Ser2448)、anti-phospho-S6(Ser235/256)、anti--phospho-4EBP1(Ser65)、anti-β-actin)孵育,4℃过夜,然后加二抗在室温下孵育1小时。用x射线胶片或数字成像系统检测印迹,蛋白质水平用密度测定法测定。
(3)蛋白免疫印迹的结果如图11所示:Dox处理后,相比于PBS组,LSC中磷酸化的mTOR、pS6、p4EBP1蛋白水平下降,而LC3-II蛋白水平明显升高,证明mTOR通路被抑制,且自噬被显著激活。而Dox@8L6 NPs可以激活mTOR通路,抑制Dox引起的LSC自噬。以上结果证实了Dox@8L6 NPs可以通过激活mTOR通路抑制LSC自噬,从而提高其对Dox的化疗敏感性。
2、Dox@8L6 NPs对AML小鼠体内治疗效果
(1)白血病细胞移植建模后第14天,将Dox@8L6 NPs与PBS或8L6 NPs或Dox作为对照连续3天静脉注射到AML小鼠体内,分别于第21天和第28天采集外周血监测白血病负荷,如图12A中所示。
(2)用流式细胞仪检测AML小鼠外周血液中GFP+白血病细胞的比例。
(3)取外周血液样本制作血液涂片。
(3)监测各组AML小鼠21天内的体重情况。
(4)第21天时用流式细胞仪检测AML小鼠骨髓中GFP+白血病细胞的比例
3、Dox@8L6 NPs对AML小鼠体内治疗结果
(1)由图12B的结果可知,Dox或Dox@8L6 NPs能够很大程度上减少外周血液中GFP+白血病细胞的比例,且Dox@8L6 NPs组表现得更加明显。
(2)图12C的血液涂片结果显示:Dox@8L6组与Dox组、PBS组相比,外周血未成熟细胞明显减少。
(3)急性髓细胞白血病是一种恶性肿瘤,作为一种消耗性疾病,可导致宿主体重下降。而图12D中的AML小鼠体重监测显示:相比于PBS组或Dox组,Dox@8L6 NPs组的体重减轻要少得多。
(4)图12E中ML小鼠骨髓中GFP+白血病细胞的比例结果显示:与PBS同时治疗组相比,8L6 NPs或Dox或Dox@8L6 NPs可以减轻骨髓白血病负担,且Dox@8L6 NPs组效果最为明显。
上述实施例中,所述的抗肿瘤药物除可以选择盐酸阿霉素外,也可以选择喜树碱、多西紫杉醇、盐酸吉西他滨、盐酸伊立替康、氟尿嘧啶或香菇多糖、姜黄素等抗肿瘤药物,亦得到相同或相近的结果。实际应用中,可根据具体的癌症类型选择相应的抗肿瘤药物与亮氨酸聚酯酰胺按照本发明的方法合成纳米递药体系,增强抗肿瘤药物的治疗效果的有效性、可控性和安全性。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
2.权利要求1中所述的基于亮氨酸的聚酯酰胺聚合物的制备方法,其特征在于:包括以下步骤:
S1.合成二元羧酸二对硝基苯基酯单体:将2~3倍于二元酰氯当量的对硝基苯酚与2~3倍于二元酰氯当量的三乙胺加入-85~-70℃的冰冻丙酮中,得到混合液A;将二元酰氯加入-85~-70℃的冰冻丙酮中,混合均匀;然后逐滴加入所述混合液A;-85~-70℃搅拌反应1~5h后转至20~30℃搅拌10~16h;
S2.合成二亮氨酸二对甲苯磺酸盐酯单体:以对甲苯磺酸一水合物作为催化剂和氨基保护剂,将L-亮氨酸、1/2~1/3L-亮氨酸当量的二元醇、2~4倍于L-亮氨酸当量的甲苯磺酸一水合物在甲苯中于110~150℃反应16~72h;
S3.将二元羧酸二对硝基苯基酯单体和二亮氨酸二对甲苯磺酸盐酯单体加入反应溶剂中,65~85℃搅拌条件下逐滴加入三乙胺作为反应催化剂,在60~80℃下加热反应24~96h,即获得所述的基于亮氨酸的聚酯酰胺;所述二元羧酸二对硝基苯基酯单体和二亮氨酸二对甲苯磺酸盐酯单体的质量比为1∶2~2∶1;
步骤S1所述二元酰氯选自乙二酰氯、丁二酰氯或辛二酰氯;
步骤S2所述二元醇选自乙二醇、1,4-丁二醇、1,6-己二醇中的一种或几种;
步骤S3所述反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺或二甲基亚砜中的一种或几种。
3.根据权利要求2所述的基于亮氨酸的聚酯酰胺聚合物的制备方法,其特征在于:
步骤S1所述反应的条件为-78℃搅拌反应2h后转至25℃搅拌10~16h;
步骤S1得到二元羧酸二对硝基苯基酯单体后将其洗涤,50~60℃真空干燥至恒重,并利用乙腈重结晶进行纯化;
步骤S2所述反应温度为120~140℃,反应时间为24~30h;
步骤S2得到二亮氨酸二对甲苯磺酸盐酯单体后,将其在70~80℃加热搅拌条件下溶解于沸水中,并在2~8℃条件下析出沉淀,重复2~4次,再于55~65℃真空干燥24~48h;
步骤S3得到聚酯酰胺聚合物后,将其倒入预冷的乙酸乙酯中析出沉淀,弃上清,将产物用甲醇复溶,复提纯除杂3~4次后,将产物于50~60℃真空干燥24~48h。
4.权利要求1中所述的聚酯酰胺聚合物在制备或作为药物载体中的应用。
5.一种基于权利要求1中所述的聚酯酰胺聚合物的纳米载体的制备方法,其特征在于:通过如下任一种方式实现:
(I)将所述聚酯酰胺聚合物溶于有机溶剂中,得到聚酯酰胺聚合物溶液;然后在搅拌条件下,将聚酯酰胺聚合物溶液滴加至含有稳定剂的水溶液中,使其自组装成纳米粒,得到聚酯酰胺聚合物药物输送载体;
或
(II)将所述聚酯酰胺聚合物和稳定剂分别溶解于有机溶剂中,得到聚酯酰胺聚合物溶液和稳定剂溶液;然后将两者混合均匀后滴加至水中,使其自组装成纳米粒,得到聚酯酰胺药物输送载体。
6.一种基于权利要求1中所述的聚酯酰胺聚合物的载药纳米体系的制备方法,其特征在于:通过如下任一种方式实现:
(I)将所述聚酯酰胺聚合物、抗肿瘤药物和稳定剂分别溶解到有机溶剂中,得到聚酯酰胺聚合物溶液、抗肿瘤药物溶液和稳定剂溶液;然后将三种溶液混合均匀后滴加至水中,使其自组装成载药纳米粒,得到聚酯酰胺载药纳米体系;
或
(II)将上述聚酯酰胺聚合物和抗肿瘤药物分别溶解到有机溶剂中,得到聚酯酰胺聚合物溶液和抗肿瘤药物溶液;然后将聚酯酰胺聚合物溶液和抗肿瘤药物溶液滴加到含有稳定剂的水溶液中,使其自组装成纳米粒,得到聚酯酰胺载药纳米体系。
7.根据权利要求6所述的制备方法,其特征在于:
方式(I)和(II)中所述的抗肿瘤药物溶液的浓度为5~50mg/mL;
方式(I)和(II)中所述的聚酯酰胺聚合物与抗肿瘤药物的质量比为9∶1~2∶1;
所述的抗肿瘤药物包括亲水性药物和疏水性药物;所述亲水性药物包括但不限于盐酸阿霉素、盐酸吉西他滨、盐酸伊立替康、氟尿嘧啶和香菇多糖;所述疏水性药物包括但不限于紫杉醇、多西紫杉醇、甲氨蝶呤、喜树碱、阿霉素和姜黄素。
8.根据权利要求5-7任一项所述的制备方法,其特征在于:
方式(I)和(II)中所述的稳定剂为聚乙烯醇、两性离子活性剂或二硬脂酰基磷脂酰乙醇胺-聚乙二醇DSPE-PEG;所述的两性离子活性剂为羧基甜菜碱或磺基甜菜碱;
方式(I)和(II)中所述的稳定剂的用量为占聚酯酰胺聚合物质量的0~75%,不包括0;
方式(I)和(II)中所述的有机溶剂为二甲基亚砜、N,N-二甲基甲酰胺和四氢呋喃中的一种或多种;
方式(I)和(II)中所述的聚酯酰胺聚合物溶液的浓度为5~50mg/mL;
方式(I)和(II)中所述的稳定剂溶液的浓度为5~50mg/mL。
9.一种纳米载体,其特征在于:通过权利要求5或8所述的制备方法制备得到。
10.一种载药纳米体系,其特征在于:通过权利要求6~8任一项所述的制备方法制备得到。
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