CN108484819B - 一类水溶性星形荧光聚合物及其纳米颗粒的制备方法 - Google Patents
一类水溶性星形荧光聚合物及其纳米颗粒的制备方法 Download PDFInfo
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- C08F220/10—Esters
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
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Abstract
本发明公开了一类水溶性星形荧光聚合物及其纳米颗粒的制备方法,其关键在于,从生物相容性良好的环糊精出发,以原子转移自由基(ATRP)聚合反应为主,获得一系列合成成本低、结构可控、水溶性良好和发射范围可调的水溶性星形荧光聚合物,采用核磁、红外光谱等表征手段对其化学结构进行系统性确定,进一步通过体外毒性、体内外成像等方面的研究,综合评估水溶性星形荧光聚合物在肿瘤前期诊断中的应用潜力。
Description
技术领域
本发明涉及荧光探针领域,具体涉及一类结构可控、毒副作用小和发射范围可调的水溶性星形荧光聚合物的制备方法及其应用。
背景技术
肿瘤的前期诊断对其后期治疗具有重要的临床应用价值,而基于荧光技术的纳米医学策略是当前最主要的途径之一。基于小分子的荧光化合物由于存在以下问题:1)毒副作用大;2)非水溶性;3)容易发生自猝灭;4)难以提取及可重复利用性低等缺点,使得小分子的荧光化合物在生命科学、医学等领域中的应用受到了限制。随着纳米技术的不断完善及发展,基于纳米技术的荧光聚合物正在被构建,该体系在小分子荧光化合物的光稳定性和反光性漂白方面有了很大的改进,这一发展使得对不同的荧光信号进行精确的检测成为可能。将小分子荧光化合物引入聚合物的侧链、链端或通过具有荧光功能单体的聚合制备的荧光聚合物,在使用过程中,可以克服小分子荧光化合物在使用上的局限性。
将具有荧光功能的单体与亲水性单体(如聚乙二醇,PEG)共聚形成的两亲性聚合物,具有有一些优势:1)水溶性良好;2)毒副作用小;3)光稳定性好;4)发射范围可调。通过这种方法可提高肿瘤的前期诊断,从而进行适时实时的治疗。CN 107353410 公开了一类两亲性聚合物及水分散醛基功能化荧光纳米胶束和制备方法,具体由疏水性的超支化共轭聚合物和亲水性的线性聚醚组成,其中超支化共轭聚合物具有荧光性质、末端带有醛基功能团。该有机聚合物纳球荧光体,其荧光体不会泄露,在功能化荧光纳米探针领域具有较好地应用前景。CN 103613692公开了一类以苝类衍生物或其类似物作为荧光核的单分子荧光聚合物胶束及其制备方法及其作为PH探针的应用,该体系光、热稳定性好、水溶性好、结构可设计。
由于现存的荧光材料的使用受限,因此,构建结构可控、水溶性良好、毒副作用小和发射范围可调的功能性荧光聚合物具有重要的意义。
发明内容
本发明的目的之一是一类水溶性星形荧光聚合物的制备方法,该制备方法合成简单,产率高。目的之二是使用所述制备方法制备的水溶性星形荧光聚合物具有结构可控、光稳定性好和发射范围可调等优点。为实现上述目的,采用以下技术方案:
水溶性星形荧光聚合物及其纳米颗粒的制备方法,包含以下步骤:
(1)MAHDO的制备,其反应式如下所示,包含以下步骤:冰浴(温度≤0℃)和氩气(Ar,2-10Pa)条件下,1,6-己二醇溶于含三乙胺的无水四氢呋喃溶液后,往其中逐滴加入甲基丙烯酰氯溶于的四氢呋喃所形成的混合液,搅拌0.5h,恢复至室温过夜,再过滤除杂, 接着用旋转蒸发仪对上述混合液中的四氢呋喃浓缩,将浓缩后的混合液进一步以乙酸乙酯、正己烷为洗脱剂过硅胶柱除去二取代产物,再用旋转蒸发仪进一步浓缩可得到6-hydroxyhexyl methacrylate (简记为MAHDO);
(2)MDye的制备,其反应式如下所示,包含以下步骤:在冰浴(温度≤0℃)和氩气(Ar,2-10Pa)条件下,含羧基的荧光染料(简记为Dye-COOH)和二环己基碳二亚胺(DCC)依次加入含有3mL二氯甲烷(DCM)的反应管中,搅拌0.5h后,将步骤(1)所制备的MAHDO与4-二甲氨基吡啶(DMAP)溶于0.5mL DCM所形成的混合液加入,搅拌0.5h后恢复至室温,待反应48h,依次用乙酸乙酯,水,盐酸,饱和食盐水萃取,收集有机相并用无水硫酸钠除水,所得到的有机相进一步用旋转蒸发仪浓缩溶剂,以DCM和甲醇为洗脱剂过硅胶柱除去未参加反应的Dye-COOH,用旋转蒸发仪浓缩所收集的溶液即为MAHDO-Dye单体(简记为MDye);
(3)ß-CD-Br的制备,其反应式如下所示,包含以下步骤:在冰浴(温度≤0℃)和氩气(Ar,2-10Pa)条件下,ß-环糊精(ß-CD)加入无水N,N-二甲基甲酰胺(DMF)中,待溶解后,用滴液漏斗逐滴加入含2-溴代异丁酰溴的DMF溶液,接着在冰浴下搅拌0.5h, 室温反应24-72h后得到引发剂(ß-CD-Br);
(4)PDye的制备,其反应式如下所示,包含以下步骤:在氩气(Ar,2-10Pa)条件下,以步骤(3)中所制备的ß-CD-Br为原子转移自由基聚合(ATRP)反应的大分子引发剂,将ß-CD-Br、乙二醇甲基丙烯酸酯(OEGMA)和步骤(1)中所制备的MDye一并溶于等体积的N,N-二甲基甲酰胺(DMF)与二甲亚砜(DMSO)形成的混合液中,加入溴化亚铜(CuBr)后,进行冷冻-解冻循环三次,再加入三-(N , N-二甲氨基乙基)胺(Me6TREN) 配体,进行冷冻解冻一次,然后在室温下避光反应24h,再加3~5mL甲醇稀释,用截留分子量(MWCO)为3500Da的透析袋在甲醇中透析24h,浓缩后得到水溶性星形荧光聚合物PDye;反应式中PDye中的x表示MDye的聚合度,x的范围为6~30,y表示OEGMA的聚合度,y的范围为2~120;
(5)水溶性星形荧光聚合物纳米颗粒的制备,包含以下步骤:称量 5mg步骤(4)所制备的PDye,溶于DMF溶液中,搅拌0.5h,用200uL移液枪逐滴加至二次水中搅拌0.5h后,用MWCO为3500Da的透析袋在水中透析24h,即得到水溶性星形荧光聚合物纳米颗粒的溶液。
进一步,所述步骤(1)中1,6-己二醇与甲基丙烯酰氯的摩尔浓度范围比为1:(0.2~1.4);TEA与THF的体积比范围为1:(8~26);乙酸乙酯、正己烷的体积比范围为1:(3.62~4.8)。
进一步,所述步骤(2)中Dye-COOH、MABHD、DCC、DMAP的摩尔比范围为1:(1~2.1) :(0.9~1.6) :(0.2~2.7);甲醇、二氯甲烷的体积比范围为1:(18~24)。
进一步,所述步骤(3) 中环糊精、二溴异丁酰溴的摩尔浓度比范围为1:(20~60) 。
进一步,所述步骤(4) 中ß-CD-Br、OEGMA、MDye、CuBr、Me6TREN的摩尔浓度比范围为1:(73~85):(73~85) :(16~24);DMF、DMSO的体积比范围为1:(0.9~1.1)。
进一步,所述步骤(5)中DMF和水的体积比范围为1:(6~500); 所述步骤(5)中透析后所得的水溶性星形荧光聚合物纳米颗粒粒径范围为1~1000nm。
主要优点:
针对小分子荧光化合物现存的水溶性差,毒副作用大等问题,本项目提出一类水溶性星形荧光聚合物的制备方法及其生物应用,该体系具有合成成本低、结构可控、水溶性良好、毒副作用小、和发射范围可调等优良特性,从而实现体内外生物成像。
附图说明
为了更加清楚的展现本发明的目的及其技术方案,本发明提供如下附图:
图1为本发明实施例1中的水溶性星形荧光聚合物的制备流程示意图。
图2、图3、图4为本发明实施例1中的水溶性星形荧光聚合物制备中间产物MAHDO、MCFD及终产物PCFD的核磁示意图。
图5为本发明实施例 1中的水溶性星形荧光聚合物的DLS和TEM示意图。
图6为本发明实施例1中的水溶性星形荧光聚合物纳米颗粒的毒性示意图。
具体实施方式
下面将结合附图,对本发明的实施例进行详细的描述
实施例1
1)MAHDO的制备:冰浴(温度≤0℃)和氩气(Ar,2-10Pa)条件下,1,6-己二醇溶于含三乙胺的无水四氢呋喃溶液后,将甲基丙烯酰氯溶于四氢呋喃所形成的混合溶液逐滴加入,搅拌0.5h,恢复至室温过夜,过滤除杂,用旋转蒸发仪浓缩溶剂,进一步以乙酸乙酯、正己烷为洗脱剂过硅胶柱除去二取代产物,用旋转蒸发仪浓缩后取适量以氘代氯仿(CDCl3)为溶剂测氢谱其结果如图2所示, 5.50ppm(标记为2)与6.05ppm(标记为1)双键峰的出现表明MAHDO的成功合成。
2)MDye的制备:在冰浴(温度≤0℃)和氩气(Ar,2-10Pa)条件下,Dye-COOH和二环己基碳二亚胺(DCC)依次加入含有3mL二氯甲烷(DCM)的反应管中,搅拌0.5h后,将MAHDO与4-二甲氨基吡啶(DMAP)溶于0.5mL DCM所形成的混合液加入,搅拌0.5h后恢复至室温,待反应48h,依次用乙酸乙酯,水,盐酸,饱和食盐水萃取,收集有机相并用无水硫酸钠除水,所得到的有机相进一步用旋转蒸发仪浓缩溶剂,以DCM和甲醇为洗脱剂过硅胶柱除去未参加反应的Dye,用旋转蒸发仪浓缩后得到单体。当Dye-COOH为5(6)羧基荧光素二乙酸酯(CFD)时,取适量产物以氘代氯仿(CDCl3)为溶剂测氢谱其结果如图3所示,双键氢(标记为1/2;6.09ppm/5.57ppm)与苯环氢(标记为10-15;8,29ppm-6.63ppm)的同时出现表明CFD与MAHDO的成功键合。
3)ß-CD-Br的制备:包含以下步骤在冰浴(温度≤0℃)和氩气(Ar,2-10Pa)条件下,ß-环糊精(CD)加入无水N,N-二甲基甲酰胺(DMF))中,待溶解后,将2-溴代异丁酰溴溶于DMF所形成的混合液用滴液漏斗逐滴加入,冰浴下搅拌0.5h, 室温反应24-72h后得到CD-Br。
4)PDye的制备:在氩气(Ar,2-10Pa)条件下,以ß-CD-Br为原子转移自由基聚合(ATRP)反应的大分子引发剂,与乙二醇甲基丙烯酸酯(OEGMA)、 MAHDO-Dye一并溶于等体积的N,N-二甲基甲酰胺(DMF)与二甲亚砜(DMSO)形成的混合液中,加入溴化亚铜(CuBr)后,冷冻-解冻循环三次,加入三-(N , N-二甲氨基乙基)胺(Me6TREN) 配体,冷冻解冻一次,在室温下避光反应24h,加3~5mL甲醇稀释,用截留分子量(MWCO)为3500Da的透析袋在甲醇中透析24h,浓缩后得终产物。当Dye-COOH为5(6)羧基荧光素二乙酸酯时,取适量产物以氘代氯仿(CDCl3)为溶剂测氢谱其结果如图4所示,OEGMA甲氧基(3.40ppm,标记为17)峰与8.02-6.94ppm(标记为10-15)苯环氢峰的出现,表明两亲性星型荧光聚合物的成功合成。
5)水溶性星形荧光聚合物纳米颗粒的制备:称量 5mg水溶性星形聚合物PDye,即PDye,溶于DMF溶液中,搅拌0.5h,用200uL移液枪逐滴加至二次水中搅拌0.5h后,用MWCO为3500Da的透析袋在水中透析24h,即得到纳米颗粒水溶液;当Dye-COOH为5(6)羧基荧光素二乙酸酯时,如图5所示,纳米胶束为大小均匀的球状,水合平均粒径为33.3nm,该尺寸的胶束尺寸易进入细胞;体外毒性如图6所示,当浓度为50ug.mL-1的胶束与宫颈癌细胞(Hela)和正常细胞的(L929)作用72h后,其对正常细胞毒副作用可以忽略,相比较,材料对癌细胞有一定的损伤。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (5)
1.一类水溶性星形荧光聚合物纳米颗粒的制备方法,其特征在于:包含以下步骤:
(1)MAHDO的制备,其反应式如下所示,包含以下步骤:温度≤0℃的冰浴和2-10Pa氩气Ar条件下,1,6-己二醇溶于含三乙胺的无水四氢呋喃溶液后,往其中逐滴加入甲基丙烯酰氯溶于的四氢呋喃所形成的混合液,搅拌0.5h,恢复至室温过夜,再过滤除杂, 接着用旋转蒸发仪对上述混合液中的四氢呋喃浓缩,将浓缩后的混合液进一步以乙酸乙酯、正己烷为洗脱剂过硅胶柱除去二取代产物,再用旋转蒸发仪进一步浓缩可得到6-hydroxyhexylmethacrylate,简记为MAHDO;
(2)MDye的制备,其反应式如下所示,包含以下步骤:在温度≤0℃的冰浴和2-10Pa氩气Ar条件下,含羧基的荧光染料Dye-COOH和 二环己基碳二亚胺DCC依次加入含有3mL二氯甲烷DCM的反应管中,搅拌0.5h后,将步骤(1)所制备的MAHDO与4-二甲氨基吡啶DMAP溶于0.5mL DCM所形成的混合液加入,搅拌0.5h后恢复至室温,待反应48h,依次用乙酸乙酯,水,盐酸,饱和食盐水萃取,收集有机相并用无水硫酸钠除水,所得到的有机相进一步用旋转蒸发仪浓缩溶剂,以DCM和甲醇为洗脱剂过硅胶柱除去未参加反应的Dye-COOH,用旋转蒸发仪浓缩所收集的溶液即为MAHDO-Dye单体,简记为MDye;
(3)ß-CD-Br的制备,其反应式如下所示,包含以下步骤:在温度≤0℃的冰浴和2-10Pa氩气Ar条件下,ß-环糊精ß-CD加入无水N,N-二甲基甲酰胺DMF中,待溶解后,用滴液漏斗逐滴加入含2-溴代异丁酰溴的DMF溶液,接着在冰浴下搅拌0.5h, 室温反应24-72h后得到引发剂ß-CD-Br;
(4)PDye的制备,其反应式如下所示,包含以下步骤:在2-10Pa氩气Ar条件下,以步骤(3)中所制备的ß-CD-Br为原子转移自由基聚合ATRP反应的大分子引发剂,将ß-CD-Br、寡聚乙二醇甲基丙烯酸酯OEGMA和步骤(2)中所制备的MDye一并溶于等体积的N,N-二甲基甲酰胺DMF与二甲亚砜DMSO形成的混合液中,加入溴化亚铜CuBr后,进行冷冻-解冻循环三次,再加入三-(N , N-二甲氨基乙基)胺Me6TREN 配体,进行冷冻解冻一次,然后在室温下避光反应24h,再加3~5mL甲醇稀释,用截留分子量MWCO为3500Da的透析袋在甲醇中透析24h,浓缩后得到水溶性星形荧光聚合物PDye;反应式中PDye中的x表示MDye的聚合度,x的范围为6~30,y表示OEGMA的聚合度,y的范围为2~120;
(5)水溶性星形荧光聚合物纳米颗粒的制备,包含以下步骤:称量 5mg步骤(4)所制备的PDye,溶于DMF溶液中,搅拌0.5h,用200uL移液枪逐滴加至二次水中搅拌0.5h后,用MWCO为3500Da的透析袋在水中透析24h,即得到水溶性星形荧光聚合物纳米颗粒的溶液。
2.根据权利要求1所述的一类水溶性星形荧光聚合物纳米颗粒的制备方法,其特征在于:所述步骤(1)中1,6-己二醇与甲基丙烯酰氯的摩尔浓度范围比为1: 0.2~1:1.4;TEA与THF的体积比范围为1: 8~1:26;乙酸乙酯、正己烷的体积比范围为1:3.62~1:4.8。
3.根据权利要求1所述的一类水溶性星形荧光聚合物纳米颗粒的制备方法,其特征在于:所述步骤(2)中Dye-COOH、MAHDO、DCC、DMAP的摩尔比范围为1:1:0.9:0.2~1:2.1:1.6:2.7;甲醇、二氯甲烷的体积比范围为1: 18~1:24。
4.根据权利要求1所述的一类水溶性星形荧光聚合物纳米颗粒的制备方法,其特征在于:所述步骤(3)中ß-环糊精、2-溴代异丁酰溴的摩尔浓度比范围为1:20~1:60 。
5.根据权利要求1所述的一类水溶性星形荧光聚合物纳米颗粒的制备方法,其特征在于:所述步骤(5)中DMF和二次水的体积比范围为1:6~1:500; 所述步骤(5)中透析后所得的水溶性星形荧光聚合物纳米颗粒粒径范围为1~1000nm。
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