CN107496918B - 一种复合纳米诊疗制剂及其制备方法 - Google Patents
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Abstract
本发明公开了一种复合纳米诊疗制剂及其制备方法。复合纳米诊疗制剂,包裹薄荷醇和IR‑780碘化物,于脂质载体中,所述载体包括磷脂、胆固醇、二肉豆蔻酰基磷脂酰乙醇胺‑二乙基三胺五乙酸‑钆。所述复合纳米诊疗制剂的粒径为90 nm‑2μm。本发明制备的集超声/光声/核磁共振成像与光热治疗功能于一体的复合纳米诊疗制剂,将多模态造影与光热治疗结合在一起,其中多模态成像为光热治疗提供了可靠的影像依据,提高了光热治疗的时间、空间选择性及准确性。另外,该复合纳米诊疗制剂因纳米尺寸所特有的EPR效应,易于在肿瘤部位富集。这样既提高了诊断效率,又降低了患者承担毒副作用的风险,加快了多模态造影与光热治疗诊疗一体化的进程。
Description
技术领域
本发明涉及生物医学领域,具体地说,涉及一种复合纳米诊疗制剂及其制备方法。
背景技术
近年来,癌症已严重威胁人类健康,对癌症的精确诊断和有效治疗是本领域长期以来研究的热点与难点。在临床实践中,肿瘤的诊断与治疗仍为两个相对独立的过程。诊断试剂与治疗用药分开使用,延长了疾病诊断-治疗的周期,造成贻误病情风险高,患者的经济负担加重。依托于纳米技术的迅猛发展,诊疗一体化制剂以其良好的生物膜穿透力、被动靶向实体瘤的高通透性和滞留效应(EPR效应)及易于多功能化等优势在生物医学领域得到广泛关注。
光热治疗技术是一种可控、非侵入式的肿瘤治疗方法。该技术利用光热材料吸收近红外光,将其转化为局部热能使肿瘤区域温度升高,从而达到杀死肿瘤细胞的目的。目前,光热材料主要包括高分子纳米材料(如聚吡咯纳米材料)、碳材料、贵金属材料及有机小分子(如吲哚菁绿、IR-780碘化物)材料。其中贵金属材料、高分子材料、碳材料类虽然有良好的光热转化能力及可修饰性,但这类材料的生物安全性及不可降解等问题仍然颇具争议;小分子染料虽然具有良好的生物相容性及可代谢等优势,但由于分子量小,存在体内循环时间短、不具备肿瘤靶向性等问题。因此,如何提高小分子染料的肿瘤靶向性、延长其体内循环时间成为该领域亟待解决的问题。
另外,单一光热转换剂无法确定治疗部位,使其在临床应用中受到极大限制。因此,为了有效确定治疗部位,实时监测治疗进程与治疗效果,将光热治疗与造影技术结合。利用造影技术高分辨率的优势,并借助具有造影增强效果的造影剂,可大大提高诊断的灵敏度及治疗效率。
超声、光声、核磁这三种成像方式是目前科学研究应用最为广泛的分子影像诊断方式,各自拥有优势特点,如超声具有实时动态成像,便携经济、时间分辨率好的特点,但是灵敏度和分辨率较低;光声成像具有深穿透的优势,但声学信号较弱;核磁灵敏度和空间分辨率大,但时间分辨率差,不能进行动态、实时成像。因此,将以上三种成像造影剂结合不仅可以提高单独成像的灵敏度,更可以通过优势互补,实现肿瘤的早期高效诊断及术中引导和术后评价,为光热治疗提供多模态图像,避免单一图像方式的局限性,可有效提高治疗效率,避免正常组织的损伤及破坏。
发明内容
本发明的目的在于针对上述诊断及治疗制剂存在的不足,提供一种生物相容性好、毒副作用小,且集超声/光声/核磁成像与光热治疗功能于一体的复合纳米诊疗制剂及其制备方法。
本发明的技术方案是通过以下方式实现的:
本发明所述的复合纳米诊疗制剂,是一种集超声/光声/核磁共振成像与光热治疗功能于一体的复合纳米诊疗制剂。
一种复合纳米诊疗制剂,包裹薄荷醇和IR-780碘化物(IR-780),于脂质载体中,所述载体包括磷脂、胆固醇、二肉豆蔻酰基磷脂酰乙醇胺-二乙基三胺五乙酸-钆(DMPE-DTPA-Gd)。所述复合纳米诊疗制剂的粒径为90 nm-2μm。
上述复合纳米诊疗制剂的制备方法,包括如下步骤:
(1)以二肉豆蔻酰基磷脂酰乙醇胺(DMPE)为载体,采用交联剂将等摩尔含量的二乙基三胺五乙酸(DTPA)分子接枝到DMPE分子上,得到DMPE-DTPA分子;
(2)将得到的DMPE-DTPA分子溶于水中,加入氯化钆(GdCl3 · 6H2O)溶液室温搅拌,透析后冻干,得到DMPE-DTPA-Gd 材料;
(3)以磷脂、胆固醇及DMPE-DTPA-Gd 作为载体原料,通过乙醇注入法包裹薄荷醇及IR-780;
(4)将混合溶液在水浴超声及探头超声条件下进行分散,结束后透析;
(5)将步骤(4)获得的产物冷冻干燥48 h后取出,4℃冰箱保存。
在上述复合纳米诊疗制剂的制备方法中,所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐或N-羟基琥珀酰亚胺。
在上述复合纳米诊疗制剂的制备方法中,步骤(3)所述磷脂为饱和磷脂或不饱和磷脂。
在上述复合纳米诊疗制剂的制备方法中,步骤(3)所述磷脂为DPPC、DSPE-PEG 、DPPG 、卵磷脂中的一种或多种的混合。
在上述复合纳米诊疗制剂的制备方法中,磷脂、胆固醇与DMPE-DTPA-Gd的质量比例范围为2-6:3:1。
在上述复合纳米诊疗制剂的制备方法中,所述薄荷醇为左旋薄荷醇、右旋薄荷醇或消旋薄荷醇中的一种或多种混合。
本发明与现有技术相比,具有如下优点:
(1)本发明制备的集超声/光声/核磁共振成像与光热治疗功能于一体的复合纳米诊疗制剂,将多模态造影与光热治疗结合在一起,其中多模态成像为光热治疗提供了可靠的影像依据,提高了光热治疗的时间、空间选择性及准确性。另外,该复合纳米诊疗制剂因纳米尺寸所特有的EPR效应,易于在肿瘤部位富集。这样既提高了诊断效率,又降低了患者承担毒副作用的风险,加快了多模态造影与光热治疗诊疗一体化的进程;
(2)本发明制备的复合纳米诊疗制剂的粒径分布均匀、性质稳定且呈单分散状态、水溶性和生物相容性良好,且磷脂包覆在薄荷醇及IR-780表面,可有效解决薄荷醇、IR780疏水性的问题;此外,DMPE-DTPA-Gd与商用钆喷酸葡胺比较,弛豫率显著提高。
(3)本发明的制备方法条件温和、可控性强、简便易行。另外,本发明制备的复合纳米诊疗制剂最终形态可为冻干粉剂,稳定性好且易于储存运输。因此,本发明在肿瘤临床诊断与治疗方面具有广阔的应用前景。
下面结合附图对本发明作进一步的说明。
附图说明
图1 为本发明实施例1中制备的复合纳米诊疗制剂的流程示意图。
图2 为本发明实施例1中制得的复合纳米诊疗制剂的透射电镜图。
图3 为本发明实施例2中制得的复合纳米诊疗制剂的核磁共振成像及弛豫率对比图。
图4 为本发明实施例3中制得的复合纳米诊疗制剂的紫外-可见光吸收光谱图。
图5 为本发明实施例4中制得的复合纳米诊疗制剂的近红外热效应图。
图6为本发明实施例5中制得的复合纳米诊疗制剂在近红外激光辐照前后的超声造影增强效果。
图7为本发明实施例6中制得的复合纳米诊疗制剂的光声成像图。
图8为本发明实施例7中制得的复合纳米诊疗制剂的粒径分布图。
图9为本发明实施例8中制备的复合纳米诊疗制剂的粒径分布图。
具体实施方式
本发明所述的复合纳米诊疗制剂,是一种集超声/光声/核磁共振成像与光热治疗功能于一体的复合纳米乳。本发明通过乙醇注入法将薄荷醇、IR-780包裹在由磷脂、胆固醇及二肉豆蔻酰基磷脂酰乙醇胺-二乙基三胺五乙酸-钆(DMPE-DTPA-Gd)的组成的脂质体中,得到性质稳定且呈单分散状态的复合纳米乳。
实施例1:
(1)DMPE-DTPA-Gd 的制备过程:
将1,2-十四酰基磷脂酰乙醇胺(DMPE)溶于氯仿,配制浓度为 0.1 mM 的溶液 4mL。将 30 μL 三乙胺逐滴加入 20 mL 1 mM 的二乙基三胺五乙酸(DTPA)溶于二甲基亚砜(DMSO)的溶液并加入交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)及N-羟基琥珀酰亚胺(NHS)。将上述两种溶液混合后在氩气的氛围内室温孵育 3 h 后,先在DMSO溶液中透析12 h (透析袋: MW 3500),再在超纯水中透析48 h。制备得到 DMPE-DTPA冻干储存。
精准称取 0.384 g DMPE-DTPA,溶于 10 mM pH=5.8 的醋酸钠缓冲溶液,再加入0.337 mM 的GdCl3 水溶液 3.1 mL,用 1 M 的NaOH 将溶液 pH 调节至 6.5,然后在 50 ℃的条件下加热 5 h。反应完全后,离心(4500 g)10 min,沉淀物用 50 mM 醋酸钠缓冲液清洗2-3次,最后离心冻干。
(2)Hybrid liposome复合纳米乳的制备过程:
将磷脂DPPC\DSPE-PEG\胆固醇\DMPE-DTPA-Gd按照质量比为5:1:3:1的比例溶解在无水乙醇溶液中,并在该溶液中加入0.5 mgIR-780。另外将10 mg薄荷醇溶于100微升无水乙醇中。
将4 mL 超纯水加入10 mL的西林瓶中,在水浴超声的条件下注入含有薄荷醇的乙醇溶液,完全分散后继续注入含有复合磷脂及IR-780的乙醇溶液,水浴超声1-2 min。另采用探头超声12 s。将样品进行透析去除乙醇。最终得到该多功能复合纳米乳(Hybridliposome)。本步制备过程如图1所示;
(3)透射电子显微镜(TEM)观察:将复合纳米乳分散液滴加到铜网上,0.3%醋酸双氧铀染色30 s,空气中干燥后使用透射电子显微镜观察复合纳米乳的形态,透射电镜照片如图2所示。从图中可以看出,该实施例制备的复合纳米乳粒径范围为70-120 nm,粒径分布较均匀。薄荷醇与IR-780可包裹在复合磷脂材料中,与图1所展示的结构相似,其中IR-780的包封率达到90%。
实施例2:
将实施例1中的Hybrid liposome复合纳米乳中的Gd含量通过ICP-MS进行检测,并将原样品按照Gd浓度为标准,稀释到一定的浓度梯度(0.01 mM/L、0.02 mM/L、0.04 mM/L、0.06 mM/L、0.08 mM/L、0.1 mM/L),并在核磁共振成像系统上实施核磁共振造影成像。结果如图3所示,随着Hybrid liposome复合纳米乳浓度的增加,T1造影增强效果越来越明显。通过定量拟合结果可以判断该复合纳米乳在浓度达到100 μg/mL时,核磁共振图像显示弛豫时间T1明显缩小。弛豫时间T1越短,弛豫率r1越大,表明材料的造影能力越强。与商用MRI造影剂钆喷酸葡胺对比,其弛豫率为商用造影剂3-4倍。由此可知,本复合纳米乳具有较高核磁共振成像灵敏度,可提供精确的诊断结果。
实施例3:
将实施例1中的Hybrid liposome复合纳米乳与游离的IR-780稀释成一定的浓度后(以蒸馏水作为空白对照),通过紫外-分光光度计检测,得到紫外-可见吸收光谱图。如图4所示,单纯的IR-780与Hybrid liposome复合纳米乳在790 nm-770这一段近红外光范围内均有很强的近红外吸收。当被薄荷醇及磷脂包覆之后,其纵向吸收峰产生了略微蓝移的现象,这是由于周围薄荷醇及磷脂球腔改变了介质的折光指数,从而引起的共振吸收波长蓝移。以上结果说明,Hybrid liposome复合纳米乳很好地保留了IR-780的光学吸收性质。
实施例4:
将实施例1中的Hybrid liposome复合纳米乳稀释成一定的浓度梯度(以含IR-780量作为浓度标准)即: 0μg/mL、20μg/mL、40μg/mL ,并以PBS缓冲溶液作为空白对照。将上述配制好的溶液(4 mL)加入比色皿中,使用波长为808 nm的二极管红外激光器在2 W/cm2的辐照功率下照射10 min,通过电子温度计每隔30 s记录实时温度。如图5所示,IR-780含量为20μg/mL的Hybrid liposome复合纳米乳10 min内升温约24 ℃,温度上升速度明显高于空白对照组(约4 ℃),随着复合纳米乳浓度的升高其升温效率随之增强。10 min内温度可升高至56℃,可有效杀死肿瘤细胞。由此可知,Hybrid liposome复合纳米乳具有较高的光热转换效率,且可在短时间内达到消融温度。
实施例5:
将实施例1中的Hybrid liposome 复合纳米乳以一定浓度加入至塑料巴氏吸管,以手术钳在液面以下夹紧密封,保证管内无气泡。在近红外激光照射(808 nm,2 W/cm2)前取超声造影图(频率:8.0,机械指数:0.4),在近红外激光照射6分钟后,样品进行超声显影并取图。如图6所示,以生理盐水作为对照,以相同的激光照射条件及超声显影条件进行操作。对照组中PBS缓冲液、薄荷醇脂质体、IR-780脂质体组照射前后均无明显超声显影增强效果,Hybrid liposome 复合纳米乳实验组在照射前无超声信号产生,当其在近红外光照射6分钟后产生明显的超声信号,其原因主要是在IR-780吸收光能并将其转化为局部热能,该热能促进薄荷醇从固态转化为液态并以气态的形式挥发,产生的气体绝热压缩系数远高于液体,因此产生显著的超声造影增强信号。
实施例6:将实施例中Hybrid liposome复合纳米乳按一定浓度稀释,并分别加入至1.5 mL离心管中,分别以生理盐水作为对照,分别通过 Endra Nexus 128小动物光声仪进行检测,如图7所示,实验组样品,在一定浓度梯度下IR-780的光声造影信号呈浓度依赖性趋势,这说明复合纳米乳中的IR-780可有效吸收光能并产生超声波从而显示为加强的光声信号,该复合纳米乳作为光声成像探针具有一定的临床应用潜质。
实施例7:
(1)同实施例1的步骤(1)。
(2)同实施例1的步骤(2),区别在于DPPG\DSPE-PEG\ 胆固醇\DMPE-DTPA-Gd按照质量比为1:1:3:1的比例溶解在无水乙醇溶液中。以及所用的薄荷醇为右旋薄荷醇。如图8所示,该方法得到的Hybrid liposome复合纳米乳粒径约为95.3 nm,粒径多分散系数(PDI)为0.09,说明复合纳米乳粒径均一、分散性好。
实施例8:
(1)同实施例1的步骤(1);
(2)同实施例1的步骤(2),区别在于卵磷脂\DSPE-PEG\胆固醇\DMPE-DTPA-Gd按照质量比为5:1:3:1的比例溶解在无水乙醇溶液中。如图9所示,该方法得到的CS-DTPA-Gd@GNR复合纳米乳粒径约为104.5nm,粒径多分散系数PDI为0.08。说明该比例磷脂制备得到的复合纳米乳粒径较均一,且具有良好的分散性。且IR-780的包封率为89%,说明该比例的复合磷脂制备的纳米乳具有较高的负载能力。
Claims (3)
1.一种复合纳米诊疗制剂,其特征在于包裹薄荷醇和IR-780碘化物的脂质载体,所述载体包括磷脂、胆固醇及二肉豆蔻酰基磷脂酰乙醇胺-二乙基三胺五乙酸-钆;
所述复合纳米诊疗制剂的粒径为90 nm-2μm;
制备方法包括如下步骤:
(1)以二肉豆蔻酰基磷脂酰乙醇胺DMPE为载体,采用交联剂将等摩尔含量的二乙基三胺五乙酸DTPA分子接枝到DMPE分子上,得到DMPE-DTPA分子;
(2)将得到的DMPE-DTPA分子溶于水中,加入氯化钆溶液室温搅拌,透析后冻干,得到DMPE-DTPA-Gd 材料;
(3)以磷脂、胆固醇及DMPE-DTPA-Gd 作为载体原料,通过乙醇注入法包裹薄荷醇及IR-780;
(4)将混合溶液在水浴超声及探头超声条件下进行分散,结束后透析;
(5)将步骤(4)获得的产物冷冻干燥48 h后取出,4℃冰箱保存;
步骤(3)所述磷脂为DPPC和DSPE-PEG的组合,DPPG和DSPE-PEG的组合或卵磷脂和DSPE-PEG的组合;
磷脂、胆固醇与DMPE-DTPA-Gd的质量比例范围为2-6:3:1。
2.根据权利要求1所述的复合纳米诊疗制剂,其特征在于,所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐或N-羟基琥珀酰亚胺。
3.根据权利要求1所述的复合纳米诊疗制剂,其特征在于,所述薄荷醇为左旋薄荷醇、右旋薄荷醇或消旋薄荷醇中的一种或多种混合。
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