CN109010274A - 一种磁热双敏型荧光胶束粒子及其制备方法 - Google Patents
一种磁热双敏型荧光胶束粒子及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种磁热双敏型荧光胶束粒子及其制备方法,本发明以N‑异丙基丙烯酰胺,N,N‑二甲基丙烯酰胺及聚乳酸等为原料合成热敏性P(NIPAM‑co‑DMAM)‑b‑PLA三嵌段聚合物,再通过同步水化、透析法将右旋糖酐‑磁性层状复合氢氧化物‑氟尿嘧啶磁性缓控释给药系统组装至热敏性胶束的核心,制备了具有磁响应和温度敏感性的磁热双敏型胶束,然后利用静电结合技术水溶性近红外CdHgTe量子点(QD)嫁接到磁热双敏型胶束层表,最终制成同时兼具近红外荧光发射、磁靶向和热敏控释性能的DMF@TRM@QD荧光胶束粒子。本发明的产品具有适宜的LCST,工艺简单、成本低廉,载药量高、水溶性好、溶胶稳定性高;胶束粒子具有理想的热敏性能、磁靶向性及荧光示踪功能,在肿瘤的靶向治疗及磁致热疗领域有特殊的应用前景。
Description
技术领域
本发明属于高分子材料及药物制剂新剂型领域,特别是涉及一种磁热双敏型荧光胶束粒子及其制备方法。
背景技术
随着人类对疾病规律认识的深化及现代制药技术的进步,药物传输的智能化命题已引起医学界的高度重视。智能给药系统能对外界的特定刺激信号给予响应,并根据刺激信号的性质、强弱自动调节药物释放,又称自我调节给药系统(Self-regulated drugdelivery systems,SRDDS)。智能给药系统应具有保护药物、局部靶向、准确控制药物释放、增强药物穿透力、自控式释放等特性;其目标是建立更高层次、药物疗效更好的靶向给药系统;研发方式通常为多重复合设计,旨在帮助药物顺利通过体内各种生理屏障,靶向到特定细胞或细胞器。
磁热敏荧光胶束是热敏给药系统发展的重要方向,其中将热敏材料与磁性药物、光学试剂的组装技术是制约热敏系统的智能化进程的关键。Fe3O4是目前国内外应用最为广泛的磁性药物载体,因此,人们针对磁性热敏胶束的研究目前主要集中于Fe3O4@胶束复合物上,但Fe3O4缺乏直接结合药物的能力,对磁性药物及胶束系统的制备极为不利。另一方面,人们针对热敏系统的荧光标记试遍了全部荧光材料——有机荧光基团、量子点及稀土氧化物,但受组装方法和荧光强度限制,用现有荧光组装技术制备的荧光胶束粒子无法满足研究热敏系统细胞转运及体内传输过程的需要。因为,目前已有的方法都是将荧光材料与Fe3O4粒子或药物分子混合包封到热敏系统内部,这会导致荧光强度降低,无法满足体内深层成像要求。
发明内容
本发明的目的旨在提供一种同时兼具近红外荧光发射、磁靶向和热敏控释性能的磁热双敏型荧光胶束粒子;
本发明的另一目的是提供上述磁热双敏型荧光胶束粒子的制备方法。
本发明采用以下技术方案予以实现上述发明目的:
一种磁热双敏型荧光胶束粒子,其特征在于该胶束是以聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸三聚体为载体,将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶组装至聚合物胶束核心,层表嫁接有水溶性近红外CdHgTe量子点的热敏性给药系统,其中N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺嵌段的亲水基团向外定向排列形成胶束壳层,疏水性碳架及聚乳酸嵌段内敛、包埋右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶形成胶束核心,具有核-壳型胶粒结构;低临界溶解温度等于或高于人体温度。
所述胶束的低临界溶解温度为42℃。
合成所述三聚体的单体为N-异丙基丙烯酰胺、N,N-二甲基丙烯酰胺和丙交酯。
上述磁热双敏型荧光胶束粒子的制备方法,其特征在于其工艺步骤为:
1)首先用2,2′-偶氮二异丁基咪盐酸盐(AMAD)作共聚引发剂,使N-异丙基丙烯酰胺(N-isopropylacrylamide,NIPAM)与N,N-二甲基丙烯酰胺(N,N-dimethylacrylamide,DMAM)通过自由基共聚生成具有端羟基的聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)二聚体[P(NIPAM-co-DMAM)二聚体],再以辛酸亚锡催化,引发该二聚体的端羟基与丙交酯开环加聚,生成双亲型聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸)三聚体[P(NIPAM-co-DMAM)-b-PLA三聚体];
2)通过透析、水化法将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体组装成磁热双敏型胶束;
3)通过水相一锅法合成水溶性近红外CdHgTe量子点;
4)利用静电结合技术将过程3)所得的水溶性近红外CdHgTe量子点嫁接到过程2)所得的磁热双敏型胶束层表。
所述二聚体的合成过程为:按照95~85:5~15的质量比将N-异丙基丙烯酰胺和N,N-二甲基丙烯酰胺混合,用有机溶剂A溶解,通N2除氧后,加入引发剂2,2′-偶氮二异丁基咪盐酸盐,在70~80℃下恒温反应10~12h,之后用过量乙醚沉淀产物,抽真空过滤、真空干燥即可,其中有机溶剂A为四氢呋喃(也可用氯仿);所述引发剂2,2′-偶氮二异丁基咪盐酸盐的用量为N-异丙基丙烯酰胺和N,N-二甲基丙烯酰胺质量的1~2%。
所述三聚体的合成过程为:将具有端羟基的P(NIPAM-co-DMAM)二聚体同丙交酯按质量比40~30:60~70混合,用有机溶剂B溶解,加入适量催化剂辛酸亚锡,通氮气除氧后,在120~140℃下恒温反应24~28h,之后用乙醚沉淀产物,真空干燥即可,其中所述有机溶剂B为无水二甲苯或甲苯。
所述右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体组装过程为:将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体用有机溶剂N,N-二甲基甲酰胺溶解,然后转移到透析袋中,用去离子水在室温、剧烈搅拌条件下透析;所述透析袋分子量截留值为8000~14000g·mol-1;所述透析时间为48h,透析过程中,前5小时中每小时更换一次去离子水,之后每12小时更换一次;所述右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体用量(以质量比计)为5~20:20。
所述利用静电结合技术将水溶性近红外CdHgTe量子点嫁接到磁热双敏型胶束层表的具体工艺过程为:
a、将水溶性近红外CdHgTe量子点和磁热双敏型胶束按照1~3:1~1的质量配比混合、研磨;
b、用无水乙醇悬浮、分散步骤a所制混合粉末,然后将混悬液超声分散、磁铁吸附分离,离心分离,再经无水乙醇洗涤后真空干燥即可,其中所述超声分散是指将混悬液放置到超声水浴中,在30~50℃温度超声振荡1~3h;所述磁分离是指用磁铁吸附超声分散后混悬液中的磁性固态物质,倾弃液相除去未结合的CdHgTe量子点;所述无水乙醇洗涤固相样品2~3次;所述真空干燥条件为50~60℃、0.085MPa。
本发明中右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶磁性缓控释给药系统(DMF)的获得可参见中国专利CN200910117371.7内容。
本发明中水溶性近红外CdHgTe量子点(QD)的获得可参见中国专利CN201810513306.5内容,具体工艺为:
a、将Cd(NO3)2用水溶解,澄清,在N2流通、室温及300rpm磁搅拌条件下除氧,然后按照摩尔比Cd2+/Hg2+=1:0.03~0.06的比例,将Hg(NO3)2溶液加入其中,分散均匀,再按照摩尔比Cd2+/巯基丙酸=1:1~2的比例加入巯基丙酸溶液,原位反应,调pH至6.0~9.0,制成Cd2+-Hg2+-巯基丙酸前体溶液;
b、按摩尔比Te/NaBH4=1:1.8~2.2称取Te粉和NaBH4固体,加水溶解,在N2流通、50℃恒温及300rpm磁搅拌条件下反应至Te粉消失,制成NaHTe浆料;
c、按摩尔比Cd2+/NaHTe=1:0.10~0.130的比例,将步骤b制得的NaHTe热浆料,加入到步骤a所制的Cd2+-Hg2+-巯基丙酸前体溶液中,在N2保护、100℃恒温及300rpm磁搅拌条件下反应至液相的荧光强度不再上升;
d、反应浆料静置、陈化后,加无水乙醇沉降、分离,弃取上清液,在常温、5000rpm条件下离心分离,所得固相样品用无水乙醇洗涤,真空干燥即可。
过程a中,所述除氧时间为30~45min,原位反应时间30~60min,调节pH用浓度为2.0mol/L NaOH溶液;过程d中,所述反应浆料静置、陈化时间50~70min,用无水乙醇洗涤2~3次,真空干燥条件为65~80℃、0.085MPa。
本发明以N-异丙基丙烯酰胺(NIPAM)与N,N-二甲基丙烯酰胺(DMAM)聚合聚乳酸(PLA)嵌段合成P(NIPAM-co-DMAM)-b-PLA[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸]热敏性聚合物(TRM),通过同步水化、透析法将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶磁性缓控释给药系统(DMF)组装至热敏性胶束的核心,制备了具有磁响应和温度敏感性的磁热双敏型胶束(P(NIPAM-co-DMAM)-b-PLA@DMF),然后利用静电结合技术水溶性近红外CdHgTe量子点(QD)嫁接到磁热双敏型胶束层表制成同时兼具近红外荧光发射、磁靶向和热敏控释性能的DMF@TRM@QD荧光胶束粒子。所制粒子能迅速转运进入细胞,受到激发后在细胞内发射红色荧光;粒子细胞内化与转运效率伴随磁场强度升高呈线性增大趋势,细胞核密度及其固缩程度也随磁场梯度呈现规律性变化,为通过改变外加磁场强度、作用方向实现对成像及化疗效果的调控提供了可能,在体内诊断成像及癌症治疗方面有特殊的应用价值。
本发明中通过亲水性NIPAM-co-DMAM嵌段的酰胺基团面向水相排列形成胶束壳层,疏水性碳架及PLA嵌段内敛、包埋DMF磁性纳米粒子形成核壳型胶束结构;具有低临界溶解温度(LCST)42℃,远远高于单独聚(N-异丙基丙烯酰)的LCST(32℃),高于人体的生理温度(37℃)而低于肿瘤组织的温度,因此,聚合物可在正常生理条件下稳定存在,在体内长期循环而不易被机体清除,但能在肿瘤加热部位沉淀下来并高度浓聚,达到热致靶向的目的,适合体内药物转运的应用要求;具有较低的临界胶束浓度(7.413μg·mL-1),有较高的溶胶稳定性和灵敏的磁响应性,胶束的透光性与粒径变化表现明显的热敏性,相变前后药物的释放呈现不同的动力学模型,药物释放速率及累积释放量随温度升高而增加。
综上,本发明是在先期解决药物的包封与靶向转运问题、研发DMF给药系统的基础上,利用DMF作为热敏性胶束的核芯,解决药物包封、靶向转运、热激释放与长效循环的矛盾;基于DMF的静电辐射将水溶性近红外量子点组装到胶束外壳,解决磁热敏胶束粒子的生物转运成像问题;同时,所合成的热敏性P(NIPAM-co-DMAM)-b-PLA载体具有适宜的LCST,制备工艺简单、成本低廉,产品的载药量高、水溶性好、溶胶稳定性高;胶束粒子具有理想的热敏性能、磁靶向性及荧光示踪功能,在肿瘤的靶向治疗及磁致热疗领域有特殊的应用前景。
附图说明
图1DMF@TRM粒子的透射电镜形貌(A)与DMF@TRM@QD粒子的荧光图像(B);
图2用DMF@TRM@QD复合物孵育1h(A)、3h(B)、5h(C)和7h(D)后MGC-803细胞的成像结果(绿色荧光代表Hoechst 33342标记的细胞核信号,红色荧光代表DMF@TRM@QD标记细胞的信号,黄色代表绿色荧光和红色荧光叠加后的荧光信号,40倍物镜观察);
图3在42℃热疗及不同梯度外磁场(磁铁数分别为0,5,10,15,20,25)干预条件下,用DMF@TRM@QD孵育7h后MGC-803细胞的共聚焦成像结果(绿色荧光代表Hoechst 33342标记的细胞核信号;红色荧光代表细胞内荧光胶束粒子的信号;黄色荧光代表绿色和红色叠加后的信号);
图4细胞中红色荧光平均光密度与外加磁场强度(T)之间的线性关系。
具体实施方式
本发明化学合成所用试剂均为常规商品试剂,生物实验所用材料均为商业产品。以下实施例旨在进一步说明本发明,而不用于限制本发明要求保护的范围。
实施例1:按以下过程合成DMF@TRM@QD胶束粒子
1)P(NIPAM-co-DMAM)-b-PLA三聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸]的制备
按质量NIPAM/DMAM=90:10的比例,分别称取0.41g的NIPAM和0.046g DMAM作合成原料,投进三颈烧瓶,加25mL新蒸四氢呋喃(THF)溶解。通N2除氧30min后,加入0.009g共聚引发剂AMAD,在80℃恒温下反应10h。产物用过量乙醚沉淀、抽真空过滤,在室温下真空干燥12h得到二聚产物:即含有端羟基的P(NIPAM-co-DMAM)-OH二聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)]。然后将上述含有端羟基的P(NIPAM-co-DMAM)-OH二聚体用过量乙醚沉淀、抽真空过滤,在室温下真空干燥12h得固相二聚体。
用按质量D,L-丙交酯/P(NIPAM-co-DMAM)-OH=67:33配比,分别称取0.1879g丙交酯和0.0909g二聚体粉末,混合后投入三颈烧瓶,用20mL无水二甲苯搅拌溶解;加2-3滴辛酸亚锡,通氮气除氧30min,在135℃下恒温反应24h,得到热敏型三聚产物:即P(NIPAM-co-DMAM)-b-PLA三聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸]。将P(NIPAM-co-DMAM)-b-PLA三聚体用乙醚沉淀,并在30℃下真空干燥48h至恒重,制得固态热敏性P(NIPAM-co-DMAM)-b-PLA三聚体。
2)磁热双敏型胶束(DMF@TRM)的制备
分别称取20mg DMF粉末和20mg P(NIPAM-co-DMAM)-b-PLA三聚体,用10mL N,N-二甲基甲酰胺溶解,然后,转移到透析袋中,以1000mL去离子水在室温、剧烈搅拌条件下透析48h。前5小时中每小时更换一次透析介质,然后,每12小时更换一次,透析2天后得到热敏胶束溶液;将上述胶束溶液取出放入干净的100mL小烧杯中,在-20℃下冷冻凝结后,再迅速转入预冷的真空冷冻干燥机升温,制成胶束冻干粉,4℃保存。
3)水溶性近红外CdHgTe量子点的制备
称取5.3211g Cd(NO3)2·4H2O固体配成100mL水溶液,取10mL加到1000mL反应器中,用900mL水稀释,在N2保护、室温及300rpm磁搅拌条件下除氧30min,再加5μL的Hg(NO3)2·4H2O饱和溶液,搅拌30min后加0.00517mol的巯基丙酸MPA,在300rpm磁搅拌条件下滴加2.0mol/L的NaOH溶液,调节浆料的pH值到7.0,制成Cd2+-Hg2+-MPA前体。
称取0.27g Te粉和0.16g NaBH4固相,投到100mL反应器中,加10mL双蒸水,在N2保护、50℃恒温及300rpm磁搅拌条件下反应至Te粉消失,制成NaHTe。将所制浆料注入Cd2+-Hg2 +-MPA前体溶液中,在N2保护、100℃恒温及300rpm磁搅拌条件下反应至液相荧光强度达到最高。浆料静置、陈化60min后加无水乙醇沉降、分离,弃取上清液,在常温、5000rpm条件下离心,用无水乙醇洗涤固相2~3次后,置于65℃、0.085MPa真空干燥箱中烘干。
4)以DMF@TRM胶束粉和水溶性近红外CdHgTe量子点为原料,通过静电复合制备DMF@TRM@QD荧光胶束粒子,工艺过程如下:
a、按质量份数QD:DMF@TRM=1:1的比例,分别称取适量水溶性近红外CdHgTe量子点和DMF@TRM胶束粉末,在玛瑙研钵中混合、研磨10~50min;
b、用无水乙醇悬浮、分散步骤a所制混合粉末,将混悬液放置到超声水浴中,在30~50℃温度超声分散2h;
c、用磁铁吸附液相中的磁性固态物质,倾弃液相除去未结合的CdHgTe量子点。重新分散、超声、磁分离,然后在常温、5000rpm条件下离心,用无水乙醇洗涤固相样品2~3次,用50~60℃、0.085MPa真空干燥箱烘干,即得最终产品。
实施例2:按以下过程合成DMF@TRM@QD胶束粒子
1)P(NIPAM-co-DMAM)-b-PLA三聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸]的制备
按质量NIPAM/DMAM=90:10的比例,分别称取0.41g的NIPAM和0.046g DMAM作合成原料,投进三颈烧瓶,加25mL新蒸THF溶解。通N2除氧30min后,加入0.009g引发剂AMAD,在80℃恒温下反应10h,得到二聚产物:含有端羟基的P(NIPAM-co-DMAM)-OH)二聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺]。然后将上述含有端羟基的P(NIPAM-co-DMAM)-OH二聚体用过量乙醚沉淀、抽真空过滤,在室温下真空干燥12h得固相二聚体。
按质量D,L-丙交酯/P(NIPAM-co-DMAM)-OH=60:40的配比,分别称取0.20g丙交酯和0.13g二聚体粉末,混合后投入三颈烧瓶,用20mL无水二甲苯搅拌溶解;加2-3滴辛酸亚锡,通氮气除氧30min,在135℃下恒温反应24h,得到热敏型三聚产物:P(NIPAM-co-DMAM)-b-PLA三聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸]。将P(NIPAM-co-DMAM)-b-PLA三聚体用乙醚沉淀,然后在30℃下真空干燥48h至恒重,制得热敏性固态P(NIPAM-co-DMAM)-b-PLA三聚体。
2)磁热双敏型胶束(DMF@TRM)的制备
称取5mg DMF粉末和20mg热敏聚合物的冻干粉,按实施例1(2)制成DMF@TRM胶束冻干粉。
3)水溶性近红外CdHgTe量子点的制备
工艺过程与实施例1(1)相同。
4)以DMF@TRM冻干粉和水溶性近红外CdHgTe量子点为原料,通过静电复合制备DMF@TRM@QD荧光胶束粒子,工艺过程如下:
a、按质量QD:DMF@TRM=1:3的比例,称取8mg水溶性近红外CdHgTe量子点和24mgDMF@TRM胶束粉末,在玛瑙研钵中混合、研磨10~50min;
b、用无水乙醇悬浮、分散步骤a所制混合粉末,将混悬液放置到超声水浴中,在30~50℃温度超声分散2h;
c、分离富集磁性固态物质,倾弃液相。将磁性固相重新分散、超声、磁分离,然后在常温、5000rpm条件下离心,用无水乙醇洗涤固相样品2~3次,用50~60℃、0.085MPa真空干燥箱烘干。
实施例3按以下过程合成DMF@TRM@QD胶束粒子
1)P(NIPAM-co-DMAM)-b-PLA三聚体[聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸]的制备
按质量NIPAM/DMAM=95:5的比例,分别称取0.60g的NIPAM和0.032g DMAM作初始合成原料,再按实施例1的技术条件和工艺过程制备三聚P(NIPAM-co-DMAM)-b-PLA高分子。
2)磁热双敏型胶束(DMF@TRM)的制备
称取15mg DMF粉末和20mg热敏聚合物的冻干粉,加10mL N,N-二甲基甲酰胺溶解,然后转移到透析袋中,以1000mL去离子水在室温、剧烈搅拌条件下透析48h。前5小时中每小时更换一次去离子水,之后每12小时更换一次,透析46h后得到热敏胶束溶液。将上述透析袋内的胶束溶液取出放入干净的100mL小烧杯,在-20℃下冷冻凝结后,迅速转入预冷的真空冷冻干燥机升华得到DMF@TRM胶束冻干粉。
3)水溶性近红外CdHgTe量子点的制备
工艺过程与实施例1(1)相同。
4)以DMF@TRM冻干粉和水溶性近红外CdHgTe量子点为原料,通过静电复合制备DMF@TRM@QD荧光胶束粒子,工艺过程如下:
a、按质量QD:DMF@TRM=1:2的比例,称取16mg水溶性近红外CdHgTe量子点和32mgDMF@TRM胶束粉末,在玛瑙研钵中混合、研磨10~50min;
b、用无水乙醇悬浮、分散步骤a所制混合粉末,将混悬液放置到超声水浴中,在30~50℃温度超声分散2h;
c、用磁铁吸附液相中的磁性固态物质,倾弃液相。将富集的固相用乙醇重新分散、超声、磁分离,然后在常温、5000rpm条件下离心,用无水乙醇洗涤固相样品2~3次,用50~60℃、0.085MPa真空干燥箱烘干。
实施效果评价
利用激光扫瞄荧光共聚焦成像技术验证DMF@TRM@QD荧光胶束粒子的细胞转运及生物成像效果,实验结果显示,DMF磁热敏胶束与生物量子点结合形成的荧光粒子能顺利进入细胞,到达细胞核区域,表现良好的荧光标记和磁、热靶向性能,在肿瘤的磁、热靶向化疗方面有一定的应用前景。
实验过程与实验结果:
(1)DMF@TRM@QD荧光胶束粒子的形貌特征
用日立HITACHI H-7560B型透射电子显微镜表征DMF@TRM胶束粒子的形貌。移取少量胶束溶液,滴两滴于铜网上,常温下自然挥干,通过电镜在80kV加速电压下观察、拍照。图1-A中,胶束粒子的核层内深色的六边形质点为DMF纳米粒子,边长58nm;外围胶束壳层厚度37nm,内腔直径119nm,胶粒直径198nm。胶束粒子的形貌表征说明六边形磁性DMF粒子对复合物胶束核壳式结构的形成起重要的导向作用。DMF磁性粒子的刚性可对胶束的腔囊型结构起到支撑作用,表面羟基可与胶束疏水基团的水化层形成氢键,使胶束的核壳式结构得到整缩、稳定和高度分散。
量子点标记能够示踪DMF热敏胶束(Thermo-responsive micelles,TRM)粒子的细胞转运轨迹。图1-B是通过100倍油镜观察、拍摄的DMF@TRM@QD复合粒子的荧光图像。所制CdHgTe量子点的尺寸在10nm以下,用488nm绿氦氖激光激发时发射红色荧光;DMF磁热敏胶束粒子室温下在水溶液中粒径约500nm,图中的粒子呈圆球形,放大测量,粒子外径约514nm,反映DMF@TRM@QD粒子的大小;DMF粒子表面富含羟基,包裹于胶束的核心,可与QD结合,所以复合粒子核心的荧光强度较高,粒子内径约243nm,代表了热敏性胶束所包载的DMF粒子集合体;胶束表面也有大量亲水性基团,外层荧光强度较弱的部分厚度约135nm,可近似反映胶束水化层的厚度。以上结果证明DMF胶束与QD成功结合形成荧光复合物。
(2)DMF@TRM@QD荧光胶束粒子的细胞转运与生物成像
将指数生长期MGC-803细胞用胰酶消化,离心制成单细胞悬液,按每孔3×105的量接种于6孔细胞培养板中培养24h至贴壁。吸弃皿内培养液上清,用PBS洗细胞3遍,然后,每孔加2mL含药培养基,常温组可分别于1h,3h,5h,7h后,取出细胞培养板,热疗组需先在42℃温箱恒温30min后再迅速转到37℃培养箱中分别于0.5h,2.5h,4.5h,6.5h,制作细胞爬片。吸弃上清,用PBS清洗3遍,加1mL Hoechst 33342(10μg·mL-1,染核剂)孵育30min,吸弃染色液,用PBS洗三遍。每孔加1mL 4%多聚甲醛固定5min,PBS洗3遍。用过火的注射器针头的针尖将盖玻片轻轻勾起并用小镊子取出,用滤纸吸干多余液体,然后,在载玻片上滴1-2滴抗荧光衰减封片剂;封片后于-20℃避光保存。共聚焦成像实验中,用40倍物镜观察细胞视野,用100倍油镜观察荧光胶束粒子;用488nm的绿氦氖激光激发QD荧光,用560-660nm的带通滤波器检测发射波长,用405nm光源激发、用430-460nm带通滤波器检测核染料Hoechst 33342荧光。
图2给出用DMF@TRM@QD胶束粒子孵育人胃癌MGC-803细胞得到的激光扫描荧光共聚焦显微图像。细胞核经Hoechst 33342染色呈绿色荧光;DMF@TRM@QD粒子进入细胞后在细胞内发射红色荧光;绿色荧光与红色荧光叠加后成为黄色。图2-A显示,胶束粒子与细胞作用1h时,细胞核形态规则、大小匀称,胞内红色荧光信号很弱,只有个别细胞核外或核的边缘出现红色荧光,说明荧光胶束粒子的内化程度较低,携载纳米药物对细胞活力状态的影响较弱。用荧光复合物孵育细胞3h时,从红色荧光分布看,进入细胞内部的荧光胶束粒子数量相对1h的数量明显增加;从细胞核形态看,胞核的直径分布开始分化,一部分细胞核膨胀、变圆,另外一部分细胞核体积收缩、显色暗淡。证明相互作用3h后,荧光胶束粒子出现在部分细胞核的中心位置,粒子内化已达到一定程度,所携载的药物释放、开始起到干预作用;胞核的膨胀可能与构成细胞器、细胞核的生物物质跟DMF粒子的综合性生化反应有关,不仅仅是由释放出来的FU药物单一作用引起,核体积收缩也许标志DMF粒子与核物质生化反应趋于终结。孵育到5h时,与图B比较看,单纯从细胞内显示的红色荧光斑点似乎并未表现明显的数目增加,但这并不证明荧光胶束粒子的细胞内化程度随时间延长没有相应增加,因为,以更小尺寸、更分散的状态进入细胞的荧光粒子,受成像角度、细胞器融合、掩蔽等影响,不一定能清晰、完整的显示出来;但图片的色调对比显示,细胞核着色的黄色成分相对图B明显增强,特别是细胞核核仁的黄色成分增加更多,证明细胞核中的红色荧光信号呈现随时间延长而增大的趋势。从细胞核的形态看,胞核密度的增大及核体积收缩的趋势,证明荧光胶束粒子在胞内的药效作用随时间延长得到相应体现。图D显示,荧光胶束粒子引起细胞核聚集状态及存活状态的变化与图C完全相似;虽然宏观可见的红色聚集态粒子的数量下降,但细胞着色程度提高,证明小尺寸、分散态荧光粒子的细胞内化程度增加。以上结果表明,本发明所制荧光磁性胶束粒子完成细胞内化动力学过程的时间小于1h,到达细胞核、引发细胞核形态明显分化的时间在给药后3h左右,细胞内的药效学行为伴随作用时间会逐渐加剧。
通过计算细胞成像图中红色荧光的平均光密度可以间接反映进入细胞的DMF@TRM@QD粒子数量。用Image-Pro Plus分析软件对正常温度下作用1h、3h、5h和7h四个实验组,统计得到红色荧光平均光密度值(见表1),对其与干预时间的长短进行相关性分析,结果符合一次方程:Dmean=0.0409t-0.0465(Dmean表示从细胞中发射出红色荧光的平均光密度,t表示荧光复合物干预时间,单位:小时),相关系数0.92,证明进入细胞的DMF@TRM@QD粒子的量随时间延伸呈现线性增长趋势。激光共聚焦显微镜结果显示,DMF@TRM@QD复合物的细胞转运及药效过程随干预时间增长而增强,被细胞摄取的纳米级荧光胶束粒子也会增多,粒子能被转运至细胞核的核仁位置,可通过与核生物物质的作用引起细胞核膨胀、收缩、固化,从而达到消除癌细胞及肿瘤组织的目的。以上结果证明,DMF@TRM@QD能够进入细胞,最终能到达细胞核区域,具有良好的细胞成像、荧光示踪及靶向磁、热化疗应用前景。
Table 1Dmean of different intervention times(n=3)
(3)DMF@TRM@QD荧光胶束粒子细胞传输效率的磁响应性
为考察外加磁场对DMF@TRM@QD细胞转运效果的影响,设置不同磁场梯度,在42℃热疗条件下干预MGC-803细胞7h,进行共聚焦成像。细胞实验时需提前在6孔细胞培养板的外板底部用无菌白胶布粘贴已消毒的磁铁;在内孔底放入洁净的盖玻片。接种指数生长期的MGC-803细胞,培养24h至贴壁。吸弃皿内培养液上清,用PBS洗细胞3遍,然后,每孔加2mL含药培养基,常温组可分别于1h,3h,5h,7h后,取出细胞培养板;热疗组需先在42℃温箱恒温30min,再迅速转到37℃培养箱中分别于0.5h,2.5h,4.5h,6.5h后取出细胞培养板,按上述(2)相同的步序制作细胞爬片,封片后进行共聚焦成像。
细胞成像结果如图3所示。图3-A显示,无磁场热疗时,能看到有红色荧光信号的细胞数量极少;图中的一个典型着色细胞,整个细胞呈现红色,放大可观察到清晰的细胞轮廓,荧光磁性胶束粒子到达细胞核的核仁,在细胞被固定、“猝灭”的瞬间,量子点荧光照亮了细胞内的所有区域。从图3-B到E,发射红色荧光的细胞数量随磁场强度升高明显增加,荧光密度相应增大。通过Image-Pro Plus软件统计不同磁场梯度(0,5,10,15,20,25个磁铁,每个微型磁子强度为5770Gauss或0.577T)的六个42℃热疗实验组的红色荧光平均光密度值(见表2),图4给出细胞内荧光密度与外加磁场梯度的线性关系,符合一次方程:Dmean=0.00536B+0.00254(Dmean表示从细胞中发射出红色荧光的平均光密度,B表示磁场强度,单位:特斯拉T,线性相关系数为0.91),证明进入细胞的DMF@TRM@QD的量随磁场梯度升高呈线性增加趋势,定量说明荧光磁性胶束粒子的细胞内磁靶向转运动力学行为。此外,图3A-F中的细胞核密度N与外加磁场梯度的关系符合N=2.6457n+20.429,R2=0.6528,表明细胞核密度N也随磁场梯度呈规律性分布。
Table 2Dmean of different magnetic field strength
以上结果说明,DMF@TRM@QD胶束粒子与细胞接触后能迅速转运进入细胞;受到激光激发时,能在细胞内发射红色荧光;外加磁场能提高粒子的细胞内化程度,并引起细胞核分布的磁响应现象。因此,DMF@TRM@QD粒子在体内诊断成像及癌症治疗方面有特殊的应用价值。
小结
DMF磁热敏胶束与生物量子点结合,可制得具有荧光标记、热敏性和磁靶向功能的DMF@TRM@QD复合粒子;DMF@TRM@QD粒子的细胞内化与转运效率伴随磁场强度升高呈线性增大趋势,细胞核密度及固缩程度也随磁场梯度呈现有规律的变化,为通过改变外加磁场强度、作用方向实现对成像及化疗效果的调控提供了可能。DMF@TRM与生物量子点的成功复合及其所表现的细胞转运效果,证明DMF@TRM@QD粒子将是一种极具发展前景和应用价值的智能型多功能化纳米系统。
Claims (13)
1.一种磁热双敏型荧光胶束粒子,其特征在于该胶束是以聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸三聚体为载体,将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶组装至聚合物胶束核心,层表嫁接有水溶性近红外CdHgTe量子点的热敏性给药系统,其中N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺嵌段的亲水基团向外定向排列形成胶束壳层,疏水性碳架及聚乳酸嵌段内敛、包埋右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶形成胶束核心,具有核-壳型胶粒结构;低临界溶解温度等于或高于人体温度。
2.按照权利要求1所述的磁热双敏型荧光胶束粒子,其特征在于所述胶束的低临界溶解温度为42℃。
3.按照权利要求1所述的磁热双敏型荧光胶束粒子,其特征在于,合成所述三聚体的单体为N-异丙基丙烯酰胺、N,N-二甲基丙烯酰胺和丙交酯。
4.一种如权利要求1至3任意一项所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于其工艺步骤为:
1)首先用2,2′-偶氮二异丁基咪盐酸盐作共聚引发剂,使N-异丙基丙烯酰胺与N,N-二甲基丙烯酰胺通过自由基共聚生成具有端羟基的聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)二聚体[P(NIPAM-co-DMAM)二聚体],再以辛酸亚锡催化,引发该二聚体的端羟基与丙交酯开环加聚,生成双亲型聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)-b-聚乳酸)三聚体[P(NIPAM-co-DMAM)-b-PLA三聚体];
2)通过透析、水化法将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体组装成磁热双敏型胶束;
3)通过水相一锅法合成水溶性近红外CdHgTe量子点;
4)利用静电结合技术将过程3)所得的水溶性近红外CdHgTe量子点嫁接到过程2)所得的磁热双敏型胶束层表。
5.按照权利要求4所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于,所述二聚体的合成过程为:按照95~85 :5~15的质量比将N-异丙基丙烯酰胺和N,N-二甲基丙烯酰胺混合,用有机溶剂A溶解,通N2除氧后,加入引发剂2,2′-偶氮二异丁基咪盐酸盐,在70~80℃下恒温反应10~12 h,之后用过量乙醚沉淀产物,抽真空过滤、真空干燥即可。
6.按照权利要求5所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于,所述有机溶剂A为四氢呋喃或氯仿;所述引发剂2,2′-偶氮二异丁基咪盐酸盐的用量为N-异丙基丙烯酰胺和N,N-二甲基丙烯酰胺质量的1~2%。
7.按照权利要求4所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述三聚体的合成过程为:将具有端羟基的聚(N-异丙基丙烯酰胺-co-N,N-二甲基丙烯酰胺)二聚体[P(NIPAM-co-DMAM)二聚体]同丙交酯按质量比40~30: 60~70混合,用有机溶剂B溶解,加入适量催化剂辛酸亚锡,通氮气除氧后,在120~140℃下恒温反应24~28 h,之后用乙醚沉淀产物,真空干燥即可。
8.按照权利要求7所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述有机溶剂B为无水二甲苯或甲苯。
9.按照权利要求4所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体组装过程为:将右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体用有机溶剂N,N-二甲基甲酰胺溶解,然后转移到透析袋中,用去离子水在室温、剧烈搅拌条件下透析。
10.按照权利要求9所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述透析袋分子量截留值为 8000~14000 g∙mol-1。
11.按照权利要求9所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述透析时间为48h,透析过程中,前5小时中每小时更换一次去离子水,之后每12小时更换一次。
12.按照权利要求4或9所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述右旋糖酐-磁性层状复合氢氧化物-氟尿嘧啶与三聚体用量以质量比计为5~20 :20。
13.按照权利要求4所述的磁热双敏型荧光胶束粒子的制备方法,其特征在于所述利用静电结合技术将水溶性近红外CdHgTe量子点嫁接到磁热双敏型胶束层表的具体工艺过程为:
a、将水溶性近红外CdHgTe量子点和磁热双敏型胶束按照1~3 :1~1的质量配比混合、研磨;
b、用无水乙醇悬浮、分散步骤a所制混合粉末,然后将混悬液超声分散、磁铁吸附分离,离心分离,再经无水乙醇洗涤后真空干燥即可。
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WO2020083300A1 (zh) * | 2018-10-24 | 2020-04-30 | 宁夏医科大学 | 一种磁热双敏型荧光胶束粒子及其制备方法 |
CN113061435A (zh) * | 2019-12-31 | 2021-07-02 | Tcl集团股份有限公司 | 一种荧光热敏复合量子点材料及其制备方法与环境温度监测led |
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CN101607091B (zh) * | 2009-07-24 | 2011-08-31 | 宁夏医科大学 | “右旋糖酐-磁性ldh-氟尿嘧啶”磁靶向缓控释三聚体的合成方法 |
CN103356484B (zh) * | 2013-07-26 | 2015-09-30 | 宁夏医科大学 | 磁性固态“右旋糖酐-mldh-氟尿嘧啶”脂质体 |
CN108743973A (zh) * | 2018-05-25 | 2018-11-06 | 宁夏医科大学 | 层状磁性荧光多功能纳米载药粒子 |
CN108578409A (zh) * | 2018-05-25 | 2018-09-28 | 宁夏医科大学 | 一种引发癌细胞核爆炸的层状磁性荧光纳米组装 |
CN109394687A (zh) * | 2018-10-24 | 2019-03-01 | 宁夏医科大学 | 双亲型三嵌段共聚物热敏性胶束及其制备方法 |
CN109010274A (zh) * | 2018-10-24 | 2018-12-18 | 宁夏医科大学 | 一种磁热双敏型荧光胶束粒子及其制备方法 |
CN109288792A (zh) * | 2018-10-24 | 2019-02-01 | 宁夏医科大学 | 载氟尿嘧啶双亲型三嵌段热敏性胶束及其制备方法 |
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2018
- 2018-10-24 CN CN201811242354.1A patent/CN109010274A/zh active Pending
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2019
- 2019-10-22 WO PCT/CN2019/112616 patent/WO2020083300A1/zh active Application Filing
- 2019-10-22 AU AU2019366621A patent/AU2019366621B2/en not_active Ceased
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WO2020083300A1 (zh) * | 2018-10-24 | 2020-04-30 | 宁夏医科大学 | 一种磁热双敏型荧光胶束粒子及其制备方法 |
CN113061435A (zh) * | 2019-12-31 | 2021-07-02 | Tcl集团股份有限公司 | 一种荧光热敏复合量子点材料及其制备方法与环境温度监测led |
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WO2020083300A1 (zh) | 2020-04-30 |
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AU2019366621A1 (en) | 2021-06-10 |
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