CN108888782A - 一种基于低频超声响应的靶向荧光脂质体及其制备方法和应用 - Google Patents
一种基于低频超声响应的靶向荧光脂质体及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种基于低频超声响应的靶向荧光脂质体及其制备方法和应用。具体提供了一种主动靶向的载药荧光声学脂质体,所述载药荧光声学脂质体由二棕榈酰磷脂酰胆碱(DPPC)、胆固醇(CHO)、聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺(DSPE‑PEG)和DSPE‑PEG‑iRGD组成的脂质双分子层膜以及包载在声学脂质体内的药物和荧光剂构成。所述药物选自甲氨蝶呤(MTX),所述荧光剂选自吲哚菁绿(ICG),所述DSPE‑PEG中的聚乙二醇(PEG)选自PEG1000‑PEG3000。
Description
技术领域
本发明属于医疗诊断治疗领域,具体涉及一种基于低频超声响应的类风湿关节炎诊疗一体化荧光脂质体及其制备方法。
背景技术
类风湿关节炎(RA)是一种以关节滑膜炎、血管翳新生、骨破坏为基本病理改变的的高度致残性自身免疫性疾病,其治疗首选药甲氨蝶呤(MTX)因有肝功损害、骨髓抑制等副作用常导致临床干预停止而发展为难治性RA。如何改进治疗方法,优化治疗手段,从而提高R A病变关节MTX药物浓度,增强MTX治疗效果,同时降低全身副作用,是RA治疗的难点问题。
随着对RA用药副作用认识的逐渐深入,以及控释技术和纳米载药技术的发展,人们逐渐将控释技术和纳米载药技术应用于RA治疗。但以往研究方案均存在各方面的弊端:例如关节腔注射混合有普通脂质微泡的MTX后利用超声微泡破泡技术联合MTX对RA动物模型进行治疗,超声微泡破泡技术可促进滑膜细胞的药物摄取,从而提高抗炎疗效。但是患者对关节腔穿刺依从性差,且RA常为多关节病变,关节腔穿刺操作更加困难,也增加了关节腔感染的几率。为了实现既能避免关节腔内穿刺注射又能增加药物局部浓聚的效果,有学者构建了载MT X的金纳米颗粒,经静脉注射后通过纳米颗粒的被动滞留效应,实现在病变部位的聚集,并联合激光促进药物释放进行RA治疗,获得了良好的效果;但治疗过程中,光热效应促进药物释放的温度难以控制、药物聚集程度无法监测,治疗效果也得不到及时反馈。
因此,非关节腔穿刺情况下实现病变关节的精准、靶向用药,并且可视化评估疗效以指导临床调整治疗方案从而实现有效的治疗干预,是优化RA治疗策略、改善RA治疗效果亟待解决的关键问题。急切需要探索一种更为无创、简便、有效的治疗RA的方法。
发明内容
针对现有技术存在的缺陷和空白,本发明所要解决的技术问题是:如何实现RA影像学引导下的超声定点药物控释、可视化靶向治疗RA及疗效监测。
声学脂质体相比于普通载药脂质微泡,既具有高载药量的特点,又具有良好的声学响应性能。而低频超声对载药声学脂质体的药物控释、促进药物的渗透具有显著优势,不仅兼有无创、穿透力强的特点,还可以实现能量聚焦,而且研究表明低频超声(LFUS)可以在声学脂质体表面产生空化效应,在提高脂质体磷脂膜的通透性增加药物释放的同时超声还可以打开组织屏障提高生物膜的通透性,增加药物向组织的渗透。声学脂质体这种载药体系兼具了载药量高、超声响应性能好、可超声控释三重优势,这些都将为进一步实现声学脂质体药物控释提供了良好的基础。
整合素ανβ3作为一种血管生成的生物标记,在活化的内皮细胞中呈高表达状态。研究表明,在类风湿关节炎的早期即可出现血管的新生,致使活化的单核细胞招募迁移至滑膜,并且增殖形成血管翳;并且有研究表明,通过αvβ3整合素抑制剂来阻断关节炎发生过程中的血管新生可以明显地降低滑膜的炎症并可增加内皮细胞的凋亡,最终抑制了类风湿关节炎的进展。而基于荧光声学脂质体的近红外成像作为一种新颖的成像模式,越来越引起人们的关注,在650nm-900nm的近红外波段,它具有组织低吸收率和低散射率的优点,大大增加了近红外成像的敏感性;同时,通过构建特异地靶向荧光载MTX声学脂质体,可以获得更高特异性的治疗效果,通过无创分子成像更加特异地探测出疾病在细胞水平的分子变化,能够更加精确的对疾病的变化过程实现监测。
本发明构建了基于低频超声响应性的靶向荧光载MTX声学脂质体,其靶向性通过iRGD多肽来实现,避免了体内引入大分子蛋白的安全隐患,同时其具备的荧光特性通过吲哚菁绿(ICG)来实现,两者的载体为载MTX的声学脂质体。本发明通过动物实验研究证实了该靶向声学脂质体在实现近红外分子荧光成像以及治疗RA疾病中的有效性,具有开发成为分子靶向治疗药物的潜力和良好前景。
本发明一个方面提供了一种主动靶向的载药荧光声学脂质体,所述载药荧光声学脂质体由二棕榈酰磷脂酰胆碱(DPPC)、胆固醇(CHO)、聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺(DSPE-PEG)和DSPE-PEG-iRGD组成的脂质双分子层膜以及包载在声学脂质体内的药物和荧光剂构成。
在本发明的技术方案中,所述药物选自甲氨蝶呤。
在本发明的技术方案中,所述荧光剂选自吲哚菁绿。
在本发明的技术方案中,所述DSPE-PEG中的PEG选自PEG1000-PEG3000。
在本发明的技术方案中,所述DPPC、CHO、DSPE-PEG和DSPE-PEG-iRGD之间的摩尔比为(45-55):(40-50):4:1。
在本发明的技术方案中,所述DSPE-PEG-iRGD通过将马来酰胺化聚乙二醇修饰的二硬脂酸磷酯酰乙醇胺(DSPE-PEG-Maleimide)与iRGD多肽于水中混合,并进行反应,最终合成DSPE-PEG-iRGD。
本发明另一个方面提供了所述的载药荧光声学脂质体的制备方法,其包括如下步骤:
1)将DPPC、CHO、DSPE-PEG和DSPE-PEG-iRGD分别溶解于有机溶剂中;
2)将上述四种成分进行混合,并将其摊成薄膜;
3)将药物和荧光剂溶解在水相中;
4)并将水相与薄膜进行水化分散得到磷脂溶液,加热超声分散并以脂质体挤出器通过100nm的核孔滤膜进行过膜,得到载药荧光声学脂质体前体溶液;
5)将步骤4)所的载药荧光声学脂质体前体溶液加入冻干保护剂,进行冻干;得到载药荧光声学脂质体。
本发明再一个方面提供了上述的载药荧光声学脂质体在制备诊断、治疗类风湿关节炎以及类风湿关节炎治疗过程中疗效监测的药物中的用途;所述载药荧光声学脂质体中的药物为MTX。
本发明的有益效果:
1)对低频超声具有良好响应性的声学脂质体具有高载药量特点,并可实现定点药物控释,促进药物的渗透。
2)使用iRGD多肽实现荧光声学脂质体的靶向性,避免了体内引入外源大分子蛋白的安全隐患。
3)使用ICG作为荧光标签,ICG是被FDA批准的临床应用染料,安全性能可靠,毒性低。
4)近红外荧光成像与超声靶向释药治疗RA这两种无创、无辐射的技术的有机结合,实现治疗与疗效评估,并指导临床调整治疗方案。
5)采用声学脂质体负载药物、连接靶向分子和荧光标签,制作工艺简单,便于工业化生产。
综上,本发明低频超声响应性靶向荧光载MTX声学脂质体能够实现对RA疾病的有效治疗和疗效监测,且具有载药率高、特异性强的特点,能够安全有效的应用于RA的临床治疗,为RA的治疗提供基础和保障。
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附图说明
图1.靶向荧光声学脂质体iELPs的制备表征及稳定性能检测。A,靶向荧光声学脂质体iELPs的示意图;B,iELPs在透射电镜下的形态;C,iELPs的粒径分布情况;D,固定超声辐照时间为120s,不同超声功率下药物释放的情况,结果显示,药物释放随着超声功率的增大而增多;E,固定超声辐照强度为2.5W/cm2,不同超声辐照时间下药物释放的情况,结果显示,药物释放随着辐照时间的延长而增多。
图2.靶向荧光声学脂质体的细胞摄取情况。为了分析靶向声学脂质体的靶向性能,通过高表达靶向性的细胞和靶点阻断实验验证了靶向声学脂质体的靶向性能,结果发现靶向声学脂质体iELPs的靶向性能远高于游离ICG和非靶向声学脂质体ELPs的靶向性能,并且通过iRGD阻断靶点后,靶向声学脂质体与细胞的结合能力明显下降,进一步表明了靶向声学脂质体的靶向特异性。
图3.靶向荧光声学脂质体iELPs的近红外成像。靶向荧光声学脂质体iELPs的近红外成像。A,静脉注射靶向荧光声学脂质体和非靶向荧光声学脂质体,于注射后的15min、1h、3h、6h、12h、24h进行成像,以观察两种声学脂质体的成像效果,结果显示靶向荧光声学脂质体组在每个时间点的成像效果远好于非靶荧光声学脂质体组;B,定量分析也显示靶向荧光声学脂质体组成像效果与非靶荧光声学脂质体组的成像效果的差异具有显著统计学差异(P<0.01)。C,静脉注射靶向荧光声学脂质体和非靶向荧光声学脂质体,于注射后的3h、24h进行成像,发现靶向荧光声学脂质体组在患病关节部位具有明显的荧光信号,在正常关节部位并没有明显的荧光信号;而非靶向荧光声学脂质体组在患病关节部位和正常关节部位均有不同程度的荧光信号,表明靶向荧光声学脂质体具有良好的成像特异性。
图4.活体评价靶向荧光声学脂质体iELPs的抗类风湿治疗效果。A,治疗期间小鼠爪部在以下组内的临床指数变化,生理盐水(G1),单纯超声辐照(1.2W/cm2,3min;G2),MTX(2mg/kg;G3),iELPs(2mg/kg of MTX;G4),ELPs(2mg/kg of MTX)后3h接受超声辐照(1.2W/cm2,3min;G5),iELPs(2mg/kg of MTX)后3h接受超声辐照(1.2W/cm2,3min;G6),误差条表示标准差(n=6)。实验组(G6)与生理盐水治疗组(G1)疗效存在显著差异,*P<0.005.B,正常小鼠(NC)和CIA小鼠在32个治疗天后关节的HE组织学染色切片(滑膜炎症,原始放大,×100).C,治疗结束后小鼠爪部的大体及影像学表现。
具体实施方式
通过以下实施例对本发明作进一步的详细描述,但应理解本发明并不受以下内容所限制。
实施例1诊疗一体化靶向荧光载MTX声学脂质体的制备
(1)DSPE-PEG-iRGD的合成:将游离iRGD和DSPE-PEG-Maleimide以摩尔比1.5:1比例置于超纯水中,在冰浴的环境下(4℃),使用转子(200r/min)的充分搅拌反应24小时,然后将反应液置于透析袋(MWCO:3500)中透析48h,以滤除杂质,最后将透析过的反应液用移液枪移至西林瓶中并置于-80℃冰箱,冷冻后将西林瓶置于冷冻干燥48h,最终合成DSPE-PEG-iRGD,冻干后置于-20℃冰箱备用。
(2)薄膜水化法与冻干法结合制备iRGD环肽修饰的低频超声响应性靶向荧光载MTX声学脂质体:将DPPC、CHO、DSPE-PEG和备用的DSPE-PEG-iRGD分别用三氯甲烷溶解,并按摩尔比(45-55):(40-50):4:1的比例用移液枪加入到试管中,在涡旋振荡器上混匀,在65℃环境下,用干燥的氮气流除去溶剂使其在试管壁上形成一层薄膜,然后将ICG和MTX用PBS混溶,随之用混溶液水化磷脂薄膜,得到一定浓度的磷脂溶液,将磷脂溶液加热到65℃,并用水浴超声振荡器将其分散至清澈透明,使用脂质体挤出器让混合液通过100nm的核孔滤膜进行过膜,添加入一定量的冻干保护剂(蔗糖,5%-10%),然后将所得脂质体置入-80所冰箱,冷冻后将西林瓶置于冷冻干燥机48h,冻干后取出,即得到iRGD环肽修饰的低频超声响应性靶向荧光载MTX声学脂质体(iELPs),冻干后置于-20置冰箱备用。
实施例2靶向荧光载MTX声学脂质体的基本表征及体外性能检测
(1)通过透射电镜和马尔文粒径分析仪对iELPs的形貌特征(图1B)和粒径(图1C)进行了测定,结果显示靶向纳米声学脂质体为(111.73±3.5)nm的球形结构;
(2)为了分析iELPs的低频超声响应性能,发明人对靶向纳米声学脂质体的超声控释能力进行了测定,结果显示,iELPs的药物释放随着超声强度以及超声辐照时间的增加而增多(图1D/1E)。
(3)为了分析靶向声学脂质体的靶向性能,发明人通过高表达αvβ3的细胞和靶点阻断实验验证了靶向声学脂质体的靶向性能,结果发现靶向声学脂质体iELPs的靶向性能远高于游离ICG和非靶向声学脂质体(ELPs)的靶向性能,并且通过iRGD阻断靶点后,靶向声学脂质体与细胞的结合能力明显下降,进一步表明了靶向声学脂质体的靶向特异性(图2)。
实施例3靶向荧光载MTX声学脂质体用于RA的引导治疗
1、RA动物模型的构建
用牛II型胶原蛋白和弗氏佐剂(包括完全弗氏佐剂和不完全弗氏佐剂)1:1的混合乳剂注射到8周龄的DBA/1J小鼠的尾根皮下,免疫过程分为两步,在第一次免疫后的第21天进行第二次免疫,以建立RA动物模型。其中,第一次免疫诱导是于DAB/1J小鼠尾根皮下注射牛II型胶原与完全弗氏佐剂混合剂,每只0.1ml;第二次免疫诱导是于不同位置DAB/1J小鼠尾根皮下注射牛II型胶原与不完全弗氏佐剂混合剂,每只0.1ml。根据关节炎指数(AI)对模型进行评估,评分标准如下:0分,无红肿;1分,足趾关节红肿;2分,足趾关节、足跖部均红肿;3分,踝关节以下均红肿;4分,包括踝关节全部红肿。每一只小鼠AI评分取四肢之和,最高16分。
2、RA动物模型试验
(1)于第二次免疫诱导后关节炎指数超过10分的RA小鼠进行近红外成像,进行分组为靶向荧光声学脂质体(iELPs)组和非靶向荧光声学脂质体(ELPs)组,静脉注射靶向荧光声学脂质体和非靶向荧光声学脂质体,于注射后的15min、1h、3h、6h、12h、24h进行成像,以观察两种声学脂质体的成像效果,结果显示靶向荧光声学脂质体组在每个时间点的成像效果远好于非靶荧光声学脂质体组(图3A);定量分析也显示靶向荧光声学脂质体组成像效果与非靶荧光声学脂质体组的成像效果的差异具有显著统计学差异(P<0.01)(图3B)。
(2)对单侧踝关节发病的小鼠进行近红外成像,以进一步验证靶向荧光声学脂质体的特异性,同样进行分组为靶向荧光声学脂质体(iELPs)组和非靶向荧光声学脂质体(ELPs)组,静脉注射靶向荧光声学脂质体和非靶向荧光声学脂质体,于注射后的3h、24h进行成像,以观察两种声学脂质体的成像效果,发现靶向荧光声学脂质体组在患病关节部位具有明显的荧光信号,在正常关节部位并没有明显的荧光信号;而非靶向荧光声学脂质体组在患病关节部位和正常关节部位均有不同程度的荧光信号,表明靶向荧光声学脂质体具有良好的成像特异性(图3C)。
(3)对发病小鼠进行治疗实验,根据超声、声学脂质体主动靶向性能、建模与否三个条件将评分达10~12分的CIA模型鼠分为6组,每组6只。具体分组情况如下:生理盐水(G1),单纯超声辐照(1.2W/cm2,3min;G2),MTX(2mg/kg;G3),iELPs(2mg/kg of MTX;G4),ELPs(2mg/kg of MTX)后3h接受超声辐照(1.2W/cm2,3min;G5),iELPs(2mg/kg of MTX)后3h接受超声辐照(1.2W/cm2,3min;G6)。采用AI评分变化,X线影像、组织病理学评价靶向声学脂质体疗效,同时进行药物安全性检测。发现载MTX靶向声学脂质体治疗的疾病模型组评分较低(平均4.12分)(图4A),X线影像及组织病理学显示关节部位组织结构清晰、柔韧度佳,且小鼠皮毛光亮、精神状态佳,对照组组织结构破坏与增生并存、结构紊乱、关节腔隙消失(图4B/4C),两组疗效比较差异具有统计学意义(P<0.01)。
在本研究之前,对于类风湿关节炎的靶向治疗和疗效监测,其他课题组也有类似的研究,如载有MTX的金纳米颗粒,经静脉注射后通过纳米颗粒的被动滞留效应,实现在病变部位的聚集,并联合激光促进药物释放进行RA治疗,获得了良好的效果,但在治疗过程中通过光热效应促进药物释放的温度难以控制、药物聚集程度无法监测,治疗效果也得不到及时反馈;而发明人所构建的靶向声学脂质体,其靶向性是通过iRGD多肽来实现的,同时具备主动靶向与被动滞留效应,可以更加精准聚集与病灶部位,减轻MTX对其他内脏器官的毒副作用。同时低频超声是一种更为安全的控释方式,更适用于关节等精细复杂的解剖结构,避免了过多能量聚集产生的热效应造成的热损伤。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种主动靶向的载药荧光声学脂质体,所述载药荧光声学脂质体由二棕榈酰磷脂酰胆碱(DPPC)、胆固醇(CHO)、聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺(DSPE-PEG)和iRGD和聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺(DSPE-PEG-iRGD)组成的脂质双分子层膜以及包载在声学脂质体内的药物和荧光剂构成。
2.根据权利要求1所述的载药荧光声学脂质体,所述药物选自甲氨蝶呤(MTX)。
3.根据权利要求1所述的载药荧光声学脂质体,所述荧光剂选自吲哚菁绿(ICG)。
4.根据权利要求1所述的载药荧光声学脂质体,所述聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺中的PEG选自PEG1000-PEG3000。
5.根据权利要求1所述的载药荧光声学脂质体,所述二棕榈酰磷脂酰胆碱、胆固醇、聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺和iRGD和聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺之间的摩尔比为(45-55):(40-50):4:1。
6.根据权利要求1所述的载药荧光声学脂质体,所述iRGD和聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺通过DSPE-PEG-马来酰亚胺与iRGD多肽于水中混合,并进行反应,最终合成iRGD和聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺。
7.根据权利要求1-6所述的载药荧光声学脂质体的制备方法,其包括如下步骤:
1)将二棕榈酰磷脂酰胆碱、胆固醇、聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺和iRGD和聚乙二醇修饰的二硬脂酰基磷脂酰乙醇胺分别溶解于有机溶剂中;
2)将上述四种成分进行混合,并将其摊成薄膜;
3)将药物和荧光剂溶解在水相中;
4)并将水相与薄膜进行水化分散得到磷脂溶液,加热超声分散并以脂质体挤出器通过100nm的核孔滤膜进行过膜,得到载药荧光声学脂质体前体溶液;
5)将步骤4)所的载药荧光声学脂质体溶液加入冻干保护剂,进行冻干;得到载药荧光声学脂质体。
8.根据权利要求1-6所述的载药荧光声学脂质体在制备诊断、治疗类风湿关节炎以及类风湿关节炎治疗过程中疗效监测的药物中的用途;所述载药荧光声学脂质体中的药物为甲氨蝶呤(MTX)。
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