CN113599520B - 一种卟啉脂质-全氟化碳纳米制剂及其制备方法和用途 - Google Patents
一种卟啉脂质-全氟化碳纳米制剂及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种卟啉脂质‑全氟化碳纳米制剂及其制备方法和用途,所制备的纳米制剂为核壳结构,包括外壳的卟啉脂质体膜层和内核的全氟化碳,可以作为光敏剂或声敏剂药物和氧气递送系统,向肿瘤同步递送光敏剂/声敏剂药物和氧气,在激光或超声辐照下,显著增强光动力治疗/声动力治疗效果,并改善肿瘤的乏氧微环境,有效抑制肿瘤的复发和转移。该体系将光敏剂/声敏剂药物共价连接在脂质分子中,并通过强疏水作用力包载全氟化碳,具有高稳定性、高载药量、高携氧量、不易泄漏等特点,在肿瘤诊疗方面具有很好的临床应用前景。
Description
技术领域
本发明属于生物医用材料领域,具体涉及一种卟啉脂质-全氟化碳纳米制剂及其制备方法和用途。
背景技术
光/声动力疗法在过去几十年的临床恶性肿瘤治疗研究中引起了越来越多的关注。通常光/声动力疗法可以在光/声敏剂联合激光/超声辐照作用下,将能量传递给O2,进而产生具有细胞毒性的活性氧(ROS),从而不可逆地破坏肿瘤细胞的蛋白质或DNA,诱导肿瘤细胞凋亡。与常规手术、化学疗法和放射疗法相比,光/声动力疗法具有无创性,可控性更高,副作用减少,耐药性可忽略不计以及精确治疗功能保留肿瘤介入和替代肿瘤消融等优点。
然而,迄今为止,光/声动力疗法的临床应用仍然受到一些无法控制和不可避免因素的影响。首先,由于实体瘤组织存在极端缺氧性质(pO2≤2.5mmHg),高度依赖O2限制了光/声动力疗法的发展。更严重的是,光/声动力治疗期间O2的消耗会进一步使得肿瘤部位的缺氧环境进一步恶化,降低光/声动力疗法效率,导致肿瘤进一步生长,并可能诱发肿瘤转移或产生治疗耐受。缺氧诱导因子-1(HIF-1)是通过在低氧应激下提供生长优势来抵抗癌细胞的细胞死亡和增殖的主要调节因子,HIF的表达增加通常与多种人类癌症中患者死亡率增加呈现正相关。在实验模型中,肿瘤细胞中HIF的过表达促进了肿瘤转移,而HIF的失活降低了肿瘤细胞的转移潜力。总之,这些临床和实验结果证明了HIF信号传导在转移性肿瘤进展中的重要作用。
为了解决这个问题,目前研究人员已开发了几种策略来缓解肿瘤的乏氧。通过高压氧治疗在临床试验中表现出有效的治疗效果,但许多不良副作用如高氧癫痫发作和气压伤是不可忽视的。近年来,许多基于生物反应的O2生成材料,如过氧化氢(H2O2)酶和二氧化锰(MnO2)纳米材料等通过与肿瘤组织中的H2O2作用而被用于提高肿瘤局部O2水平。但是,它们的O2生成能力都受到细胞内H2O2浓度低的限制。相比之下,全氟化碳(PFC)具有高生物相容性和很好的氧气溶解性,有望作为人工血液,用于递送氧气来缓解肿瘤乏氧,但常规递送系统中全氟化碳的负载能力通常比较低,因此需要外部刺激来触发氧气释放。另一方面,大多数传统的光/声敏剂存在载药量不足或所包埋光/声敏药物自身聚集严重,以及循环过程中药物提前泄漏等问题,导致疗效受到限制。因此,需要具有高效率和生物安全性的、更有效的光/声敏药物和氧气递送系统来提高光/声动力治疗效果,缓解肿瘤乏氧,抑制肿瘤复发和转移。
发明内容
针对现有技术中存在的问题,本发明设计了一种新型卟啉脂质-全氟化碳纳米制剂,以开发具有荧光/CT成像功能的氧气自供应光/声动力治疗系统。在该体系中,卟啉基团共价连接于脂质可以实现高载药量,有效避免卟啉光/声敏剂在体循环期间的泄漏。在氧气吸附饱和后该体系能够有效递送氧气到肿瘤组织并逐渐释放氧气,从而使肿瘤局部的乏氧状况得到显著改善,而不需要任何外部刺激。这种氧气自供应体系在提高肿瘤光/声动力效率的同时,还可抑制肿瘤复发和防止肿瘤细胞转移。
本发明的目的之一是提供一种高效的氧气递送系统,用于缓解肿瘤组织的乏氧微环境。
本发明的目的之二是提供一种高效的光/声动力治疗系统,该系统可实现光/声敏药物和氧气的高载药量,并实现同步输送肿瘤组织,实现光/声动力治疗的氧气自供应,显著提高光/声动力疗效。
本发明的目的之三是提供一种微创治疗方法,在消灭原位肿瘤的同时,还可有效避免其复发和转移。
本发明的目的是通过以下技术方案实现的:
一种卟啉脂质-全氟化碳纳米制剂,其特征在于:所述纳米制剂为核壳结构的纳米粒子,其结构包括外壳的卟啉脂质体膜层和内核的全氟化碳;所述的卟啉脂质体膜层由卟啉脂质或卟啉脂质与磷脂成分组成,其中,卟啉脂质的摩尔百分比可以是5%-100%,磷脂成分的摩尔百分比可以是0-95%。
进一步的,所述全氟化碳包括但不限于全氟丙烷、全氟戊烷、全氟己烷、全氟溴辛烷、全氟冠醚中的任意一种或者几种之间的组合。
进一步的,所述卟啉脂质具有如下式I所示的结构,包含亲水头部、疏水性碳链和卟啉基团三部分。其中,R1、R2为C6~C18烷基;R3为羟基或聚乙二醇(PEG链),其中PEG链可通过酰胺键或者酯键连接到卟啉脂质分子中;a,b=2或3;X为NH或O,即光敏剂和脂质的连接方式是酯键或酰胺键。
进一步的,具有光敏功能的卟啉基团包括但不限于四苯基卟啉、血卟啉、原卟啉、焦脱镁叶绿酸-a、紫红素-18、维替泊芬、二氢卟吩e6。
卟啉脂质的合成步骤参考中国专利(ZL.201010222238.0),在本发明中将连接不同卟啉基团所得的卟啉脂质对应为卟啉脂质A(四苯基卟啉)、卟啉脂质B(血卟啉)、卟啉脂质C(原卟啉)、卟啉脂质D(焦脱镁叶绿酸-a)、卟啉脂质E(紫红素-18)、卟啉脂质F(维替泊芬)、卟啉脂质G(二氢卟吩e6)。
进一步的,所述的磷脂成分可以包括如下任意一种或多种:可以延长血液循环时间的DSPE-mPEG2000(二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000)和DSPE-mPEG5000(二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000),可以用来修饰靶向分子的DSPE-Maleimide(二硬脂酰磷脂酰乙醇胺改性马来酰亚胺)、DSPE-mPEG2000-Maleimide(二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺)、二棕榈酰磷脂酰胆碱(DPPC)和二硬脂酰磷脂酰胆碱(DSPC)。
进一步的,所述纳米制剂的表面可以修饰肿瘤靶向分子,肿瘤靶向分子包括可以靶向肿瘤的抗体、多肽、适配体、叶酸中的任意一种。
进一步的,所述纳米制剂包载的全氟化碳可以携载氧气,所述纳米制剂携带氧气的能力是通过全氟化碳对氧气的溶解实现的。在15~40℃、氧气分压为0~101325Pa的环境中,每毫升全氟化碳可以溶解0~2毫升氧气。
本发明所述的卟啉脂质-全氟化碳纳米制剂通过如下步骤制备:
1)将卟啉脂质或卟啉脂质和磷脂成分溶解在三氯甲烷和甲醇的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在30-60℃水浴中水化10-30min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟化碳,同时使用探头超声分散5-15min,即可得到分散均匀的卟啉脂质-全氟化碳纳米制剂,可进一步使用离心方法去除未包载的全氟化碳和卟啉脂质或者磷脂。
本发明的卟啉脂质-全氟化碳纳米制剂可用于缓解肿瘤乏氧,增强肿瘤的光/声动力学治疗效果。
本发明的有益效果:
本发明的优点之一是卟啉脂质中光/声敏药物以共价键连接,全氟化碳与卟啉和疏水脂质碳链通过疏水作用力包载,所得到的载体系统具有高载药量和高稳定性,可有效避免或者减少药物在血液循环中的提前泄漏,降低毒副作用。
本发明的优点之二是卟啉脂质-全氟化碳纳米制剂可以同时将光/声敏药物和氧气靶向递送到肿瘤部位。光/声敏药物的靶向递送可以增加光/声动力疗效,氧气的靶向递送可以显著改善肿瘤乏氧,两者之间的结合可以有效抑制肿瘤生长,防治肿瘤转移。
本发明的优点之三是卟啉脂质-全氟化碳纳米制剂具有增强荧光/CT成像的功能,可以实现影像引导下的光/声动力治疗。
附图说明
图1.本发明所述的一种卟啉脂质-全氟化碳纳米制剂的结构示意图。
图2.实施例1制备的卟啉脂质-全氟溴辛烷纳米制剂的透射电镜图像,图像说明该纳米制剂为球形结构。
图3.实施例6中不同浓度O2@PFOB@PGL纳米粒子在脱氧水中释放氧气的能力比较。
图4.实施例7中PGL纳米粒、O2@PFOB@PGL纳米粒以及PBS在不同光照时间下产生活性氧能力比较。
图5.实施例8中通过钙黄绿素AM/PI共染色法检测PGL纳米粒、O2@PFOB@PGL纳米粒以及PBS在光照和未光照下的荧光显微镜图片。
图6.实施例9中HT-29肿瘤在PGL纳米粒、O2@PFOB@PGL纳米粒以及PBS在光照和未光照下的光动力MTT杀伤效果比较。
图7.实施例10中光动力治疗过程中各组肿瘤区域乏氧情况的体外检测比较。
图8.实施例11中光动力治疗中各组肿瘤体积随时间的变化图。
具体实施方式
本发明具体实施方式所列举的实施例只用于说明本发明,并不限制本发明的内容。
实施例1
本发明所述的一种卟啉脂质-全氟溴辛烷纳米制剂的制备方法包括以下步骤:
1)将含四苯基卟啉光/声敏基团的卟啉脂质A(其中R1和R2均为C16烷基;R3为羟基;a=2,b=2;X为NH)(10mmol,PGL脂质)溶解在三氯甲烷和甲醇(体积比9:1)的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在60℃水浴中水化30min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟溴辛烷(350μL,PFOB),同时使用探头超声分散5min,即可得到分散均匀的卟啉脂质-全氟溴辛烷纳米制剂,可进一步使用离心方法(1000rpm)去除未包载的全氟溴辛烷和卟啉脂质。
结果如图2所示,所得到的纳米粒子尺寸均匀,大小约为20-40nm。
实施例2
本发明所述的一种卟啉脂质-全氟己烷纳米制剂的制备方法包括以下步骤:
1)将含血卟啉光/声敏基团的卟啉脂质B(其中R1和R2均为C12烷基;R3为聚乙二醇;a=2,b=2;X为O)(9.5mmol)和DSPE-mPEG2000(0.5mmol)溶解在三氯甲烷和甲醇(体积比9:1)的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在45℃水浴中水化10min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟己烷(330μL),同时使用探头超声分散10min,即可得到分散均匀的卟啉脂质-全氟己烷纳米制剂,可进一步使用离心方法(1500rpm)去除未包载的全氟己烷和卟啉脂质以及磷脂。
实施例3
本发明所述的一种卟啉脂质-全氟己烷纳米制剂的制备方法包括以下步骤:
1)将含原卟啉光/声敏基团的卟啉脂质C(其中R1和R2均为C18烷基;R3为聚乙二醇;a=3,b=3;X为O)(0.5mmol)、DPPC(9.0mmol)和DSPE-Maleimide(0.5mmol)溶解在三氯甲烷和甲醇(体积比9:1)的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在50℃水浴中水化20min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟己烷(352μL),同时使用探头超声分散10min,即可得到分散均匀的卟啉脂质-全氟己烷纳米制剂,可进一步使用离心方法(1500rpm)去除未包载的全氟己烷和卟啉脂质以及磷脂。
4)向纳米制剂中加入0.6mmol肿瘤靶向分子巯基化PEG叶酸,搅拌24小时,使巯基化PEG叶酸与Maleimide偶联。
5)为了除去没有连接到载体表面的巯基化PEG叶酸,使用排阻色谱柱Sephadex G-50除去游离的巯基化PEG叶酸。
实施例4
本发明所述的一种卟啉脂质-全氟戊烷纳米制剂的制备方法包括以下步骤:
1)将含焦脱镁叶绿酸-a光/声敏基团的卟啉脂质D(其中R1和R2均为C8烷基;R3为羟基;a=3,b=3;X为O)(5.0mmol)、DSPC(4.5mmol)和DSPE-mPEG5000(0.5mmol)溶解在三氯甲烷和甲醇(体积比9:1)的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在40℃水浴中水化15min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟戊烷(340μL),同时使用探头超声分散5min,即可得到分散均匀的卟啉脂质-全氟戊烷纳米制剂,可进一步使用离心方法(1500rpm)去除未包载的全氟戊烷和卟啉脂质以及磷脂。
实施例5
本发明所述的一种卟啉脂质-全氟冠醚纳米制剂的制备方法包括以下步骤:
1)将含二氢卟吩e6光/声敏基团的卟啉脂质G(其中R1和R2均为C6烷基;R3为羟基;a=2,b=2;X为O)(7.0mmol)、DSPC(2.5mmol)和DSPE-mPEG2000-Maleimide(0.5mmol)溶解在三氯甲烷和甲醇(体积比9:1)的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在60℃水浴中水化25min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟冠醚(360μL),同时使用探头超声分散15min,即可得到分散均匀的卟啉脂质-全氟冠醚纳米制剂,可进一步使用离心方法(2500rpm)去除未包载的全氟冠醚和卟啉脂质以及磷脂。
4)向纳米制剂中加入0.6mmol肿瘤靶向分子RGD多肽,搅拌24小时,使RGD多肽的巯基与Maleimide偶联。
5)为了除去没有连接到载体表面的RGD多肽,使用排阻色谱柱Sephadex G-50除去游离的RGD多肽。
实施例6
以实施例1所得到的卟啉脂质-全氟溴辛烷纳米系统(PFOB@PGL)进行载氧和氧气释放研究。
将PFOB@PGL置于无菌氧气室中,O2流速为5L/min,持续15分钟以使PFOB@PGL中的全氟溴辛烷达到氧饱和度,命名为O2@PFOB@PGL。为了测量氧气释放量,将便携式溶解氧仪置于15mL脱氧水中,然后加入5mL O2@PFOB@PGL溶液,对溶液中的氧浓度进行实时探测。每个浓度进行三次重复(n=3)。PFOB@PGL中每1mL PFOB的氧负载能力计算如下:ΔO2(增强的氧浓度)×最终溶液的体积/PFOB的体积。
结果如图3所示,当O2@PFOB@PGL加入脱氧水中,可以清楚地检测出脱氧水中氧含量在显著的增加,随着PFOB含量的增加相应地表现出更高的氧释放能力,表明PFOB@PGL纳米粒子具有优异的氧气负载和在乏氧环境时的逐渐氧释放能力。
实施例7
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行体外单线态氧的检测。
为了研究O2@PFOB@PGL的单线态氧产生能力,使用SOSG作为单线态氧(1O2)的探针。首先,将0.342mL样品和0.38mL 100μM SOSG在黑色96孔板(Costar)中混合(液面高度刚好与96孔板上边缘一致),光照(650nm,200mW)适当时间(0-3分钟),通过使用多功能酶标仪(Synergy HT,Bio-Tek)测量荧光强度(在504nm激发并在525nm处测量)来定量氧化的SOSG荧光。所有操作均在避光条件下进行,每组的实验一式三份进行。
实验结果如图4所示,该探针SOSG可以在1O2的存在下被氧化,导致荧光强度增加。图中显示了520nm处SOSG的荧光强度与照射时间的函数变化图,可以看到在3分钟内,随着照射时间的延长,SOSG荧光逐渐增加。在O2@PFOB@PGL的情况下,SOSG荧光强度随照射时间的急剧增加证实了载氧之后的O2@PFOB@PGL可产生更多的1O2。相反,在单独PGL纳米粒子存在下,SOSG荧光强度增加得慢得多。与之形成鲜明对比的是,PBS中的SOSG在光照下几乎没有发生变化,进一步证实SOSG的氧化是由单线态氧引起的,而不是由光照引起的。
实施例8
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行细胞光动力治疗效果定性评估。
为了评估各种处理后的细胞活力,进行钙黄绿素-AM(钙黄绿素乙酰氧基甲酯,Calcein-AM)和PI(碘化丙锭)共染色以检测光动力治疗对癌细胞的作用。将HT-29细胞(1×105/孔)接种于6孔细胞培养箱中24h,然后用PGL纳米粒子或O2@PFOB@PGL纳米粒子(含等量4μM PGL)加入培养12h。激光照射(650nm,0.2w/cm2)180s后,继续孵育24h,在每个孔中加入Calcein-AM/PI染色剂,孵育30分钟,然后用PBS洗涤3次。最后,在荧光显微镜下观察细胞,分别在绿色通道和红色通道中记录钙黄绿素和碘化丙啶的成像。
实验结果如图5所示。仅在用PGL+光或O2@PFOB@PGL+光处理的组中观察到细胞死亡(因PI可以透过死细胞而发射红色荧光);与不含PFOB的PGL+光照组相比,O2@PFOB@PGL+光照显示出更明显的细胞死亡,这进一步证明了PFOB载氧并在光动力治疗过程中供氧可显著增强光动力治疗效果;而单独的光照处理或单独孵育PGL或O2@PFOB@PGL处理没有观察到细胞死亡。
实施例9
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行细胞光动力治疗效果定量评估。
将HT-29细胞接种于96孔板(4×104个/孔)。培养24小时后,加入不同浓度的PGL纳米粒子或O2@PFOB@PGL纳米粒子。为了评估缺氧环境中的细胞毒性,将细胞保存在一个三重气体的细胞培养箱中,培养环境为5%O2、90%N2和5%CO2。约4h后,对每个孔进行光照10min(650nm,0.2w/cm2)。然后用新鲜培养基清洗,再进行24小时孵育。将MTT溶液加入每个孔中,在37℃和5%的CO2下孵育2h。最后用酶标仪测试在490nm处的吸光度,以评估细胞活性。
实验结果如图6所示。对于没有加入PGL或O2@PFOB@PGL的组(浓度为0μM),在不同处理中未观察到细胞活性的降低。随后进行光照,随着PGL或O2@PFOB@PGL浓度的增加,HT-29细胞的活性逐渐降低。此外,当PGL浓度低于0.25μM时,O2@PFOB@PGL组显示出比PGL更高的光动力杀伤效果,这是由于PFOB中所载入的氧气有效增强了1O2的产生能力,该结果与实施例8中钙黄绿素-AM/PI染色的结果一致。
实施例10
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行体内检测肿瘤乏氧程度的评估。
使用4-5周龄的BALB/c小鼠,在小鼠的右后腿构建HT-29皮下瘤模型,待肿瘤体积增长到100mm3左右时,经尾静脉向小鼠注射200μL PBS,PGL纳米粒子(2mg/mL),O2@PFOB@PGL纳米粒子(2mg/mL)。给药后20h,对肿瘤组织进行光照3min,随后向小鼠腹腔注射60mg/kg的哌莫硝唑盐酸盐(溶于PBS中)。腹腔注射30分钟后,处死小鼠,取出肿瘤,浸入O.C.T.包埋剂,迅速在液氮中冷冻,使用冰冻切片机切成4μm薄片,使用HypoxyprobeTM-1plus乏氧检测试剂盒对冰冻切片进行免疫荧光染色,使用10μg/mL DAPI对细胞核进行染色15分钟后,0.1%PBST漂洗,用抗荧光衰减封片剂封片,使用激光共聚焦显微镜观察和拍摄荧光图像。
实验结果如图7所示,PBS组和PGL纳米粒组肿瘤缺氧明显,而O2@PFOB@PGL组可有效缓解瘤内乏氧。光动力治疗后,PGL纳米粒子+光照组肿瘤缺氧程度较PBS组更严重。与此相反,O2@PFOB@PGL+光照在治疗后肿瘤组织几乎没有乏氧区,这进一步证实O2@PFOB@PGL体系向递送氧气并缓解肿瘤乏氧的能力。
实施例11
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行动物光动力治疗效果考察。
将携带HT-29肿瘤的小鼠随机分成6组:PBS,PBS+光照,PGL,PGL+光照,O2@PFOB@PGL和O2@PFOB@PGL+光照。静脉注射24小时后,不同组用650nm激光照射(200mW)处理20分钟。
实验结果如图8所示,单独激光照射处理显示出很少的抗肿瘤作用,表明所使用的激光剂量的安全性。同时,没有激光照射的PGL和O2@PFOB@PGL均未显示肿瘤抑制,表明这些纳米粒子对肿瘤细胞没有细胞毒性。与之形成鲜明对比的是,由于在激光照射下增强的光动力治疗效应,具有PGL+光照和O2@PFOB@PGL+光照组显示出显著的肿瘤抑制。更重要的是,O2@PFOB@PGL+光照组显示出比PGL+光照组更好的肿瘤治疗效果,这进一步证实O2@PFOB@PGL纳米体系可递送O2减缓肿瘤乏氧,并增强光动力治疗效果以完全消除肿瘤。
实施例12
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行细胞声动力治疗效果定性评估。
为了评估各种处理后的细胞活力,进行钙黄绿素-AM(钙黄绿素乙酰氧基甲酯,Calcein-AM)和PI(碘化丙锭)共染色以检测声动力治疗对癌细胞的作用。将HT-29细胞(1×105/孔)接种于6孔细胞培养箱中24h,然后用PGL纳米粒子或O2@PFOB@PGL纳米粒子(含等量4μM PGL)加入培养12h。超声辐照(频率:1MHz,强度:1.5W/cm2,占空比:50%)300s后,继续孵育24h,在每个孔中加入Calcein-AM/PI染色剂,孵育30分钟,然后用PBS洗涤3次。最后,在荧光显微镜下观察细胞,分别在绿色通道和红色通道中记录钙黄绿素和碘化丙啶的成像。
结果显示,仅在用PGL+超声或O2@PFOB@PGL+超声处理的组中观察到细胞死亡(因PI可以透过死细胞而发射红色荧光);与不含PFOB的PGL+超声组相比,O2@PFOB@PGL+超声辐照显示出更明显的细胞死亡,这进一步证明了PFOB载氧并在声动力治疗过程中供氧可显著增强声动力治疗效果;而单独的超声处理或单独孵育PGL或O2@PFOB@PGL处理没有观察到细胞死亡。
实施例13
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行细胞声动力治疗效果定量评估。
将HT-29细胞接种于96孔板(4×104个/孔)。培养24小时后,加入不同浓度的PGL纳米粒子或O2@PFOB@PGL纳米粒子。为了评估缺氧环境中的细胞毒性,将细胞保存在一个三重气体的细胞培养箱中,培养环境为5%O2、90%N2和5%CO2。约4h后,对每个孔进行光照300s(频率:1MHz,强度:1.5W/cm2,占空比:50%)。然后用新鲜培养基清洗,再进行24小时孵育。将MTT溶液加入每个孔中,在37℃和5%的CO2下孵育2h。最后用酶标仪测试在490nm处的吸光度,以评估细胞活性。
结果显示,对于没有加入PGL或O2@PFOB@PGL的组(浓度为0μM),在不同处理中未观察到细胞活性的降低。随后进行超声辐照,随着PGL或O2@PFOB@PGL浓度的增加,HT-29细胞的活性逐渐降低。此外,当PGL浓度低于0.5μM时,O2@PFOB@PGL组显示出比PGL更高的声动力杀伤效果,这是由于PFOB中所载入的氧气有效增强了1O2的产生能力。
实施例14
以实施例6中所得到的载氧卟啉脂质-全氟溴辛烷纳米系统(O2@PFOB@PGL)进行动物声动力治疗效果考察。
将携带HT-29肿瘤的小鼠随机分成6组:PBS,PBS+超声辐照,PGL,PGL+超声辐照,O2@PFOB@PGL和O2@PFOB@PGL+超声辐照。静脉注射24小时后,不同组用超声(频率:1MHz,强度:1.5W/cm2,占空比:50%)处理900s,进一步考察各组肿瘤体积变化,具体过程同实施例11。
结果显示,单独超声辐照处理显示出很少的抗肿瘤作用,表明所使用的超声辐照剂量的安全性。同时,没有超声辐照的PGL和O2@PFOB@PGL均未显示肿瘤抑制,表明这些纳米粒子对肿瘤细胞没有细胞毒性。与之形成鲜明对比的是,由于在超声辐照下增强的声动力治疗效应,PGL+超声辐照和O2@PFOB@PGL+超声辐照组显示出显著的肿瘤抑制效果。更重要的是,O2@PFOB@PGL+超声辐照组显示出比PGL+超声辐照组更好的肿瘤治疗效果,这进一步证实O2@PFOB@PGL纳米体系可递送O2减缓肿瘤乏氧,并增强声动力治疗效果以完全消除肿瘤。
Claims (7)
2.根据权利要求1所述的卟啉脂质-全氟化碳纳米制剂,其特征在于:所述卟啉基团是光敏功能基团,选自四苯基卟啉、血卟啉、原卟啉、焦脱镁叶绿酸-a、紫红素-18、维替泊芬、二氢卟吩e6。
3.根据权利要求1所述的卟啉脂质-全氟化碳纳米制剂,其特征在于:所述纳米制剂的表面修饰有肿瘤靶向分子,所述肿瘤靶向分子选自可以靶向肿瘤的抗体、多肽、适配体、叶酸中的任意一种。
4.根据权利要求1所述的卟啉脂质-全氟化碳纳米制剂,其特征在于:所述纳米制剂包载的全氟化碳携载有氧气。
5.权利要求1~4任一所述的卟啉脂质-全氟化碳纳米制剂的制备方法,包括以下步骤:
1)将卟啉脂质溶解在三氯甲烷和甲醇的混合溶剂中,减压旋干液体形成薄膜,真空干燥过夜;
2)加入超纯水,在30-60℃水浴中水化10-30min后,使用超声仪在冰浴条件下超声处理,使得脂质均匀分散到水溶液中;
3)在冰浴条件下往步骤2)中溶液逐滴滴加全氟化碳,同时使用探头超声分散5-15min,即可得到分散均匀的卟啉脂质-全氟化碳纳米制剂。
6.根据权利要求5所述的制备方法,其特征在于:步骤3)进一步使用离心方法去除未包载的全氟化碳、卟啉脂质。
7.权利要求1~4任一所述的卟啉脂质-全氟化碳纳米制剂在制备缓解肿瘤乏氧和/或增强肿瘤的光/声动力学治疗效果的药物中应用。
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