CN104945630B - 生物可降解超分子嵌段共聚物及共聚物胶束的制备方法 - Google Patents
生物可降解超分子嵌段共聚物及共聚物胶束的制备方法 Download PDFInfo
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Abstract
本发明涉及疏水性药物的包裹与靶向释放领域,旨在提供一种生物可降解超分子嵌段共聚物及共聚物胶束的制备方法。该超分子嵌段共聚物由A组分与B组分构成:A组分为单端二氨基三嗪官能化的聚乙二醇,分子量在1~10kDa之间;B组分为单端胸腺嘧啶或尿嘧啶的聚内酯,分子量在0.7~8kDa之间;A、B两组分的嵌段间通过二氨基三嗪与胸腺嘧啶或尿嘧啶基团间的三重氢键作用相连。本发明所制备超分子嵌段共聚物将具有较高的稳定性。对温度、盐离子、pH值等外界环境敏感,其结构与性能易调,有望用于可控释放等领域。该方法具有良好的通用性,除制备嘧啶端官能化的聚内酯外,也可用于制备嘧啶端官能化的聚环状碳酸酯。
Description
技术领域
本发明涉及疏水性药物的包裹与靶向释放领域,特别涉及一种具有环境敏感性的、生物可降解超分子嵌段共聚物及共聚物胶束的制备方法。
背景技术
两亲性嵌段共聚物是指同一大分子中既含有亲水嵌段又含有疏水嵌段的嵌段共聚物。两亲性嵌段共聚物的不同嵌段通常是热力学不相容的,可以发生微相分离,但由于嵌段间共价键相互作用,相分离被限制在微观尺寸范围内。在选择性溶剂中,由于不同嵌段间溶解性的差异,当共聚物浓度超过临界胶束浓度(CMC)时,两亲性嵌段共聚物可形成核壳胶束等自组装结构,由于其可增溶和包载疏水性药物,所以两亲性嵌段共聚物胶束在药物包覆、缓释领域具有良好应用空间。从生物医用安全性的角度考虑,通常要求两亲性嵌段共聚物具有生物可降解性和生物相容性。生物可降解聚酯,例如聚丙交酯(PLA)、聚乙交酯(PGA)、聚戊内酯(PVL)、聚(己内酯)(PCL)等,是一类典型的疏水性高分子,具有可生物再生与降解、生物相容的优点,聚乙二醇(PEG)是最常用的亲水性高分子,具有良好的生物相容性,所以生物可降解聚酯与PEG的两亲性嵌段共聚物是制备药物载体的理想材料。
通常的两亲性嵌段共聚物中疏水、亲水链段间通过共价键相连,当亲水、疏水嵌段间通过非共价键作用(如多重氢键、金属配位键等)相连时,可形成超分子嵌段共聚物,其在水中可进一步自组装成超分子胶束。由于非共价键对外界环境(如温度、pH值、盐离子浓度等)敏感,超分子胶束通常具有环境敏感性,在药物的可控、靶向释放领域具有潜在应用空间。核酸互补碱基之间可形成多重氢键,该多重氢键具有刺激响应性,对温度、pH值、盐离子浓度敏感。文献(Wang D等,Biomacromolecules 2011,12,1370-1379)已报导基于腺嘌呤-尿嘧啶核酸碱基对之间氢键作用所形成的超分子嵌段共聚物在水中可自组装成超分子胶束,在酸性条件下,碱基上氨基发生质子化,导致氢键断裂,因此该超分子胶束在pH<5时将发生团聚,这可实现药物的可控释放。但由于腺嘌呤-尿嘧啶之间的相互作用为二重氢键,其氢键作用较弱,这可能导致其嵌段共聚物和胶束的稳定性较差。因此,采用更强的氢键作用(如三重、四重氢键)将是构筑稳定性高、环境敏感的超分子嵌段共聚物与胶束的有效方法。
发明内容
本发明要解决的技术问题是,克服现有技术中的不足,提供一种简单的制备生物可降解超分子嵌段共聚物及共聚物胶束的制备方法。
为解决技术问题,本发明的解决方案是:
提供一种生物可降解超分子嵌段共聚物,该超分子嵌段共聚物由A组分与B组分构成:A组分为单端二氨基三嗪官能化的聚乙二醇(PEG-DAT),分子量在1~10kDa之间;B组分为单端胸腺嘧啶或尿嘧啶的聚内酯,分子量在0.7~8kDa之间;A、B两组分的嵌段间通过二氨基三嗪与胸腺嘧啶或尿嘧啶基团间的三重氢键作用相连,所形成的三重氢键键合超分子嵌段共聚物的结构如下所示:
式中:R1为H或CH3,R2为H、CH3或C2H5;m为聚内酯的重复单元数,在6~168之间;n为聚乙二醇的重复单元数,在22~227之间;x为CH2的个数,在0~4之间。
本发明进一步提供了前述生物可降解超分子嵌段共聚物胶束的制备方法,具体步骤如下:
(1)A组分的制备
将聚乙二醇单甲醚(PEG)、氰乙酸、4-二甲氨基吡啶(DMAP)和二环己基碳二亚胺(DCC)置于干燥的烧瓶中,然后加入干燥的二氯甲烷,在冰水浴中反应24小时;反应结束后,过滤除去不溶物,滤液经浓缩后滴加至冰乙醚中沉淀,过滤、干燥后,得到聚乙二醇氰乙酸酯;再将聚乙二醇氰乙酸酯、二氰二胺、KOH和二甲基亚砜(DMSO)置于烧瓶中,氩气保护下在90℃反应24小时,反应结束后将反应混合物滴入冰乙醚中沉淀,过滤、干燥后,得到单端二氨基三嗪官能化的聚乙二醇;
其中,聚乙二醇单甲醚∶氰乙酸∶4-二甲氨基吡啶∶二环己基碳二亚胺的摩尔比为1∶1.2∶0.04∶1.2,二氯甲烷的质量是聚乙二醇单甲醚的15倍;聚乙二醇氰乙酸酯∶二氰二胺∶KOH的摩尔比为1∶2.5∶1,二甲基亚砜的质量是聚乙二醇氰乙酸酯的2.5倍;
(2)B组分的制备
将引发剂、内酯单体、辛酸亚锡和有机溶剂置于干燥的烧瓶中,氩气保护下,在80~140℃反应4~16小时;反应结束后,将反应混合物滴入冰乙醚中沉淀,过滤、干燥后,得到单端胸腺嘧啶或尿嘧啶官能化的聚内酯;
其中,内酯单体与引发剂的摩尔比为4~70∶1,辛酸亚锡占内酯单体质量的0.05%~1%,有机溶剂质量为内酯单体的2~4倍;所述引发剂是1-(2-羟乙基)胸腺嘧啶或1-(2-羟乙基)尿嘧啶,有机溶剂是干燥的二甲基甲酰胺(DMF)或二甲基亚砜(DMSO);
(3)将等摩尔的A组分和B组分加入四氢呋喃(THF)中溶解,并使聚合物混合液的总浓度为5g/L;将聚合物混合液滴加至3倍于四氢呋喃体积的去离子水中,搅拌;然后将混合溶液装入截留分子量为3500Da的透析袋中,在去离子水中透析24小时,冷冻干燥后,得到生物可降解超分子嵌段共聚物胶束。
本发明中,所述内酯单体是:丙交酯(LA)、乙交酯(GA)、戊内酯(VL)、ε-己内酯(εCL)或γ-己内酯(γCL)中的任意一种。
与现有技术相比,本发明具有以下优点:
(1)本发明采用二氨基三嗪与胸腺嘧啶或尿嘧啶基团间的三重氢键作用构筑超分子嵌段共聚物,三重氢键作用力强,所制备超分子嵌段共聚物将具有较高的稳定性。
(2)由于本发明所选择三重氢键具有可逆性,对外界环境敏感,所以所制备超分子嵌段共聚物及其胶束对温度、盐离子、pH值等外界环境敏感,其结构与性能易调,有望用于可控释放等领域。
(3)本发明以羟基官能化的胸腺嘧啶或尿嘧啶为引发剂,通过开环聚合制备嘧啶端官能化的聚内酯,该方法具有良好的通用性,除制备嘧啶端官能化的聚内酯外,也可用于制备嘧啶端官能化的聚环状碳酸酯。
附图说明
图1为实施例1中超分子胶束的粒径分布图。
图2为实施例1在磷酸缓冲液中的载药释放曲线。
具体实施方式
下面结合附图与具体实施方式对本发明作进一步详细描述:
PEG单甲醚、辛酸亚锡购自Sigma-Aldrich公司;丙交酯(LA)购自普拉克公司;GA、VL、εCL、γCL购自百灵威公司;LA和GA在乙酸乙酯中重结晶纯化,VL、εCL、γCL经减压蒸馏纯化;DMF、DMSO使用氢化钙干燥48小时后减压蒸馏后使用。
1-(2-羟乙基)胸腺嘧啶采用文献(Ueda等,Makromol.Chem.1968,120,13-20)方法制备,1-(2-羟乙基)尿嘧啶采用文献(Gi等,J.Org.Chem.1997,62,88-92)方法制备。
步骤一:PEG-DAT的制备
PEG-DAT参照文献(Bayer F等,Langmuir 2011,27,12851-12858)方法制备。将PEG(10g,2mmol)、氰乙酸(0.2g,2.4mmol)、DMAP(9.8mg,0.08mmol)、DCC(0.49g,2.4mmol)置于干燥的100mL烧瓶中,加入50mL干燥二氯甲烷,在冰水浴中反应24小时。反应结束后,过滤除去不溶物,滤液经浓缩后滴至冰乙醚中沉淀,过滤,干燥,得到PEG氰乙酸酯。
将PEG氰乙酸酯(1mmol)、二氰二胺(0.21g,2.5mmol)、KOH(0.056g,1mmol)、10mLDMSO置于100mL烧瓶中,氩气保护下在90℃反应24小时,反应结束后将反应混合物滴入冰乙醚中沉淀,过滤,干燥得到PEG-DAT。
所合成PEG-DAT中PEG的分子量在1~10kDa之间,表示为PEGx-DAT,其中x表示PEG链段的数均分子量。
步骤二:单端胸腺嘧啶或尿嘧啶官能化聚内酯的制备
将一定比例的1-(2-羟乙基)胸腺嘧啶或1-(2-羟乙基)尿嘧啶、内酯单体、辛酸亚锡(催化剂)、干燥DMF或DMSO置于干燥的烧瓶中,氩气保护下,在80~140℃反应4~48小时。反应结束后,将反应混合物滴入冰乙醚中沉淀,过滤,干燥,得到单端胸腺嘧啶或尿嘧啶官能化的聚内酯。聚内酯的分子量可通过改变单体与引发剂的比例进行调控。单端胸腺嘧啶或尿嘧啶官能化聚内酯制备中原料的种类、投料比、反应条件、所得聚合物分子量如表1所示。
表1单端胸腺嘧啶或尿嘧啶官能化聚内酯的制备条件与分子量
注:在聚合物代号中,下标表示聚合物的分子量,THY和URA分布表示端基为胸腺嘧啶和尿嘧啶。
NMR测试:利用核磁共振(Bruker公司,400MHz)测试聚合物的1H NMR谱图,进而计算其数均分子量(Mn)。测试温度为室温,溶剂为氘代氯仿,化学位移(δ)由溶剂峰校正。分子量计算说明:对于PLA,通过比较端羟基相邻的叔碳上的氢(δ=4.3)与主链中叔碳上的氢(δ=5.1)的峰面积比计算聚合度和分子量;对于PVL、PεCL和PγCL,利用端羟基相邻的仲碳上的氢(δ=3.6)与酯基相连的亚甲基上氢(δ=4.1)的峰面积比计算其聚合度和分子量。
步骤三:超分子胶束的制备
在实施例1~7中,将等摩尔PEG-DAT与单端胸腺嘧啶(或尿嘧啶)官能化聚内酯溶于四氢呋喃(THF)中,聚合物的总浓度5g/L,将聚合物溶液滴加至3倍于THF体积的去离子水中,搅拌。然后将混合溶液装入透析袋(截留分子量=3500Da)中,在去离子水中透析24小时,冷冻干燥得到超分子胶束。
在对比例1中,采用由共价键键合的PLA-b-PEG制备胶束。将100mg的嵌段共聚物溶解于10mL THF中,待嵌段共聚物完全溶解,在搅拌条件下将嵌段共聚物溶液滴加至3倍于THF体积的去离子水中。在室温搅拌条件下,使THF完全挥发,得到无色透明的胶束溶液。
以抗癌药物阿霉素(DOX)为例,对胶束进行载药和药物缓释实验。将10mg的DOX·HCl、三倍摩尔当量的三乙胺溶解于3mL DMSO中,避光搅拌,待DOX完全溶解后,将DMSO溶液倒入10mL含有100mg的超分子聚合物的THF溶液中,避光搅拌。然后将混合溶液滴加至20mL去离子水中,搅拌均匀后,将上述混合溶液转移至透析袋(截留分子量=3500)中,在去离子水中透析24h。透析后将胶束溶液冷冻干燥,得到载药胶束。取5mg冻干的载药胶束溶解于2mL DMSO中,用紫外-可见光分光光度计测定溶液在485nm波长处的吸光度,基于标准曲线计算胶束的载药量(DLC)。
利用动态光散射和表面张力法测定超分子胶束的流体力学直径(Dh)和临界胶束浓度(CMC)。具体测试方法如下:
DLS分析:利用DLS(Zetasizer 3000HAS,马尔文公司)测试超分子嵌段共聚物胶束的Dh。
CMC测定:采用表面张力法测定了超分子嵌段共聚物的CMC。将样品配制成不同浓度(1×10-4~1.0g/L)的水溶液,然后在20℃条件下用表面张力仪测试溶液的表面张力。CMC由表面张力与样品浓度关系图求得。
表2实施例1~7中超分子胶束的A、B组分、Dh、CMC与DLC
当超分子嵌段共聚物分散于水溶液中时,当浓度小于CMC时,其表面张力随浓度增大而降低;当浓度大于CMC时,随浓度变化其表面张力基本不变。这说明超分子嵌段共聚物在水溶液中发生胶束化,形成核壳胶束。如表2和图1所示,超分子胶束的Dh在59~246nm之间,可通过亲、疏水链段的长度进行调控,DLS所测粒径与由透射电子显微镜(TEM)所观测的粒径相吻合。与相似共聚组成的共价键键合共聚物(对比例1)相比,超分子胶束的粒径较大,这可为药物的包覆提供便利。
如表2所示,超分子嵌段共聚物胶束对DOX药物具有较高的包载率,其DLC值在1.3%~15.5%之间,可通过亲、疏水链段的长度进行调控。与相似共聚组成的共价键键合的共聚物(对比例1)相比,超分子胶束的载药量较大。
所制备超分子胶束具有环境敏感特性。对于实施例1的样品,当pH至由7降低至1时,其粒径由199.2nm增加至865.4nm;当氯化钠盐离子浓度由0M增加至0.25M时,其粒径由199.2nm增加至1000nm以上。说明在酸性环境及盐离子存在下,超分子胶束将发生聚集。但对于对比例1的共价键键合嵌段共聚物胶束,胶束粒径不随pH与盐离子浓度的变化而变化。
超分子胶束对包裹在胶束核内的药物具有缓释效果。取5mg冻干的载药胶束分散于2mL生理盐水中,待胶束分散均匀,将溶液转移至透析袋(截留分子量=3500)中,对10mLPBS缓冲溶液(pH=7.4,50mM或100mM)进行透析。透析袋外的缓冲溶液经一定的时间间隔进行更换,同时利用紫外分光光度计测试缓冲溶液中的DOX浓度,进而计算累计释放量。图2为实施例1样品的药物释放曲线。在最初的2小时内,DOX的释放速度较快,累计释放量达到17%。此后,随着时间的延长,DOX的释放速率逐渐变慢,释放30个小时后,累计释放量达到50%。
最后,需要注意的是,以上列举的仅是本发明的具体实施例。显然,本发明不限于以上实施例,还可以有很多变形。本领域的普通技术人员能从本发明公开的内容中直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (3)
1.一种生物可降解超分子嵌段共聚物,其特征在于,该超分子嵌段共聚物由A组分与B组分构成:A组分为单端二氨基三嗪官能化的聚乙二醇,分子量在1~10kDa之间;B组分为单端胸腺嘧啶或尿嘧啶的聚内酯,分子量在0.7~8kDa之间;A、B两组分的嵌段间通过二氨基三嗪与胸腺嘧啶或尿嘧啶基团间的三重氢键作用相连,所形成的三重氢键键合超分子嵌段共聚物的结构如下所示:
式中:R1为H或CH3,R2为H、CH3或C2H5;m是聚内酯的重复单元数,取值范围为6~168;n是聚乙二醇的重复单元数,取值范围为22~227;x是CH2的个数,取值范围为0~4。
2.权利要求1所述生物可降解超分子嵌段共聚物胶束的制备方法,其特征在于,具体步骤如下:
(1)A组分的制备
将聚乙二醇单甲醚、氰乙酸、4-二甲氨基吡啶和二环己基碳二亚胺置于干燥的烧瓶中,然后加入干燥的二氯甲烷,在冰水浴中反应24小时;反应结束后,过滤除去不溶物,滤液经浓缩后滴加至冰乙醚中沉淀,过滤、干燥后,得到聚乙二醇氰乙酸酯;再将聚乙二醇氰乙酸酯、二氰二胺、KOH和二甲基亚砜置于烧瓶中,氩气保护下在90℃反应24小时,反应结束后将反应混合物滴入冰乙醚中沉淀,过滤、干燥后,得到单端二氨基三嗪官能化的聚乙二醇;
其中,聚乙二醇单甲醚∶氰乙酸∶4-二甲氨基吡啶∶二环己基碳二亚胺的摩尔比为1∶1.2∶0.04∶1.2,二氯甲烷的质量是聚乙二醇单甲醚的15倍;聚乙二醇氰乙酸酯∶二氰二胺∶KOH的摩尔比为1∶2.5∶1,二甲基亚砜的质量是聚乙二醇氰乙酸酯的2.5倍;
(2)B组分的制备
将引发剂、内酯单体、辛酸亚锡和有机溶剂置于干燥的烧瓶中,氩气保护下,在80~140℃反应4~16小时;反应结束后,将反应混合物滴入冰乙醚中沉淀,过滤、干燥后,得到单端胸腺嘧啶或尿嘧啶官能化的聚内酯;
其中,内酯单体与引发剂的摩尔比为4~70∶1,辛酸亚锡占内酯单体质量的0.05%~1%,有机溶剂质量为内酯单体的2~4倍;所述引发剂是1-(2-羟乙基)胸腺嘧啶或1-(2-羟乙基)尿嘧啶,有机溶剂是干燥的二甲基甲酰胺或二甲基亚砜;
(3)将等摩尔的A组分和B组分加入四氢呋喃中溶解,并使聚合物混合液的总浓度为5g/L;将聚合物混合液滴加至3倍于四氢呋喃体积的去离子水中,搅拌;然后将混合溶液装入截留分子量为3500Da的透析袋中,在去离子水中透析24小时,冷冻干燥后,得到生物可降解超分子嵌段共聚物胶束。
3.根据权利要求2所述的方法,其特征在于,所述内酯单体是:戊内酯、ε-己内酯或γ-己内酯中的任意一种。
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