CN114796156A - 一种线粒体靶向的光热/化疗协同的纳米药物递送粒子及其制备方法和应用 - Google Patents
一种线粒体靶向的光热/化疗协同的纳米药物递送粒子及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种线粒体靶向的光热/化疗协同的纳米药物递送粒子及其制备方法和应用,所述纳米药物递送粒子通过Au‑S以及脂质的互插作用成功的将lipid‑DOX引入到了体系中,并实现了PTX的负载。体外相关实验表明,该体系具有明显的线粒体靶向性,并能刺激内源性活性氧(ROS)的产生,实现基于线粒体途径的高效肿瘤抑制作用。体内抗肿瘤实验结果表明,该体系能够通过光热‑药物‑氧化应激的协同作用产生优异的抗肿瘤效果。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种线粒体靶向的光热/化疗协同的纳米药物递送粒子及其制备方法和应用。
背景技术
随着社会的发展和人们生活方式的改变,恶性肿瘤已成为威胁人类健康的主要疾病。目前,化疗、手术和放疗仍是肿瘤治疗的重要手段,但是这些治疗手段都存在这很多弊端,例如创伤大、副作用大、容易复发和周期长等。纳米载体药物传递系统(NDDS)以其特异性的靶向性,更好的肿瘤部位渗透性和滞留性(EPR效应),改善化疗药物的药效学和药代动力学以及绕过生物屏障的能力在肿瘤治疗中显示出优势。此外,NDDS结构的多样性为研究人员对其进行修饰和改性提供了无限的可能性,从而能够具有不同的活性和靶向部分,增加其潜在的应用,其中一个非常重要的应用就是为药物组合提供了一种巧妙的“一体式”策略。“一体式”策略是将两种或两种以上的化疗药物共同负载在同一个NDDS中,利用NDDS特异性的靶向性和EPR效应,可以更有效地发挥联合用药的协同效应。
与单一药物相比,两种或两种以上无叠加毒性、无交叉耐药的化疗药物联合使用,可以作用于细胞中的不同位点、调节不同的信号通路,有效克服肿瘤细胞异质性和适应性导致的对单一靶点药物不敏感和耐药的问题,从而最大限度地发挥治疗效果。例如Yin课题组将水飞蓟宾和紫杉醇联用,共负载于一两亲聚合物纳米载药体系中,其中水飞蓟宾能够抑制细胞增殖、诱导凋亡和抗血管生成活性并增加肿瘤细胞对紫杉醇等化疗药物敏感,两者联用可以大大提高体系的抗肿瘤疗效。
有报道称,靶向不同的亚细胞器可以达到更好的抗肿瘤效果。线粒体是细胞的动力源,在调节肿瘤细胞的基本功能,包括细胞的新陈代谢、三磷酸腺苷(ATP)的生成、细胞凋亡和活性氧(ROS)的产生等方面起着核心作用。有文献表明,线粒体靶向策略通过调节细胞代谢、ATP消耗、ROS生成和促进细胞凋亡,从而更有效地逆转耐药,提高抗肿瘤疗效。例如陈的团队制备了一种具有肿瘤微环境响应性电荷反转和线粒体靶向功能的多糖纳米粒,并负载姜黄素,并引入了亲脂性阳离子物质—小檗碱衍生物作为一种新型的线粒体靶向物质,提高纳米颗粒的线粒体靶向能力;当该体系到达线粒体时,可以引起线粒体膜电位降低和细胞色素C的释放,最终导致凋亡途径的激活。因此,构建基于线粒体的抗肿瘤平台具有重要意义。除此之外,一些化疗药物除了能够通过其固有的治疗模式发挥抗肿瘤疗效,还能够刺激线粒体产生ROS杀死肿瘤细胞,例如铂类药物能通过与DNA相互作用造成DNA损伤,表现出良好的细胞毒作用,同时还能增加细胞内ROS水平以诱导细胞凋亡。
联合用药大大提高了抗癌效果,但单一治疗也存在疗效差、肿瘤复发等问题。这些问题的存在促进了多模式肿瘤治疗系统的发展,如化疗、光热治疗(PTT)、光动力治疗(PDT)等的综合应用。PTT是一种通过光热剂将近红外光转化为热能实现局部非侵入性杀灭癌细胞的有效方法,自问世以来一直受到研究人员的广泛关注。由于具有较强的近红外吸收能力、良好的生物相容性和光热稳定性,金纳米棒(AuNR)作为一种有效的光热剂已被广泛应用于肿瘤的治疗。除此之外,有文献报道在热应激和PPT过程中可以产生ROS,诱导细胞凋亡。
发明内容
为解决上述问题,本发明设计了一种基于联合给药和线粒体靶向策略的多模式一体化系统用于药物-光热-氧化应激的协同治疗。提供了一种纳米药物递送粒子,并同时提供了其制备方法和应用。
本发明一方面提供了一种纳米药物递送粒子,其为为以金纳米棒为核,以阿霉素脂质体为壳的核壳结构,其粒径长度为45~50nm,宽度为12~14nm。
进一步的,所述核壳结构通过金-硫键及脂质体互插作用构建。
更进一步的,还包括负载于所述脂质体层的纳米药物紫杉醇。
更进一步的,所述紫杉醇的包封率为15%~20%,优选为15%。
本发明另一方面提供了一种上述纳米药物递送粒子的制备方法,具体包括如下步骤:
1)将DPSE-PEG2000-NHS和DOX溶解DMSO中,并加入三乙胺,搅拌后透析,在透析袋中加入DMSO使样品完全溶解,重复6次后,再透析;
2)将CTAB溶液加入到HAuCl4溶液中搅拌均匀,然后滴加冰冷的NaBH4溶液,至溶液变成棕黄色,继续搅拌,备用;在的圆底烧瓶中加入CTAB溶液,搅拌下加入AgNO3溶液,HAuCl4溶液以及超纯水,搅拌均匀,随后滴加抗坏血酸溶液,在此过程中溶液逐渐变为无色,最后向体系中加入种子溶液,避光搅拌,溶液变为紫红色,离心,并用超纯水洗涤两次,加水制备成溶液;
3)将所述步骤1)得到的产物,DSPE-PEG2000-SH,CHOL,PTX溶解在氯仿/甲醇混合溶液中,旋转蒸发成薄膜,40℃真空干燥过夜,用所述步骤2)得到的溶液将所述薄膜超声溶解,搅拌后,超纯水洗涤一次,得到产物。
进一步的,所述步骤1)中,所述DPSE-PEG2000-NHS和所述DOX的质量比为67.5:20。
进一步的,其特征在于所述步骤2)中,所述种子溶液制备步骤中所述CTAB和所述HAuCl4和所述NaBH4的摩尔比为2:0.005:0.1;所述AuNR溶液制备中,所述HAuCl4与所述CTAB和和AgNO3的摩尔比为23:20:4;所述HAuCl4与所述种子的摩尔比为46:1。
进一步的,其特征在于所述步骤3)中,所述lipid-DOX、SPE-PEG2000-SH、CHOL和PTX和所述AuNR的质量比为2.0:0.185:0.25:0.5:1。
进一步的,所述纳米载体递送系统中PTX负载率为15%(w/w,150mg/g),DOX负载率为20%(w/w,200mg/g)。
本发明再一方面还提供了上述纳米药物递送粒子在制备黑色素瘤化疗药物、光热联合治疗药物方面的应用。
本发明的有益效果如下:
本发明通过Au-S键将DSPE-PEG2000-SH与AuNR连接,然后通过疏水作用实现了DSPE-PEG2000-DOX和DSPE-PEG2000-SH的互插式自组装,与此同时,PTX也通过疏水作用被负载到脂质层的疏水腔中,由于DSPE-PEG2000-DOX的线粒体靶向作用,可以将整个载药粒子靶向至线粒体。当PTX作用于线粒体时可以诱导细胞凋亡,提高抗肿瘤疗效;而且,DOX和PTX均可以刺激细胞产生氧化应激;此外,AuNR的光照治疗的效果在直接杀死肿瘤细胞的同时,还能够促进ROS的产生,最终实现了药物-光热-氧化应激协同治疗的给药模式。
本发明合成了长径比约为3.8的AuNR,该纳米粒子形状均一、分散性良好。AuNR能够吸收纵向波长附近的光,并将吸收的光能转化为热能,从而产生光热效果,我们所合成的AuNR纵向吸收波长在808nm附近,因此在该波长的NIR照射下该体系具有高效的光热转换效率。
本发明利用Au-S键将脂质连接在了AuNR的表面,然后利用脂质的疏水性互插在AuNR的周围包裹了一层脂质层,并将化疗药物PTX负载其中。体外释放行为可以看出所负载的药物呈现贯序释放,即lipid-DOX的释放速度明显快于PTX,说明形成脂质层结构稳定,能够作为药物载体用于药物递送。
本发明所提供的药物递送粒子实现DOC和PTX协同递送。
本发明所提供的药物递送粒子实现了线粒体靶向,光热、化疗协同治疗平台。
附图说明
图1为TEM图片,其中:(A)为AuNR,(B)为AuNR-lipid-DOX/PTX(scale bar=50nm)。
图2为水合粒径和Zeta电位,其中:(A)为AuNR,(B)为AuNR-lipid-DOX/PTX。
图3 AuNR-lipid-DOX/PTX体系的光热曲线;其中,(A)不同浓度的AuNR-lipid-DOX/PTX在2.0W/cm2近红外激光照射下的光热曲线;(B)AuNR-lipid-DOX/PTX(100ug/mL)在不同近红外激光强度照射下的光热曲线;(C)AuNR-lipid-DOX/PTX(100ug/mL)在重复近红外激光照射下的光热曲线。
图4 AuNR-lipid-DOX/PTX体系的药物释放曲线。
图5 B16 AuNR-lipid-DOX/PTX的细胞摄取及线粒体靶向结果。
图6细胞与AuNR-lipid-DOX/PTX体系各组别共孵育24h后的细胞存活率。
图7 AuNR-lipid-DOX/PTX体系细胞内刺激ROS产生的结果;其中,(A)利用DCFH-DA作为ROS探针检测ROS的激光共聚焦;(B)DCF的荧光强度统计;(C)不同AuNR-lipid-DOX/PTX组DCF归一化荧光强度。(1)AuNR-PTX,(2)AuNR-lipid-DOX,(3)lipid-DOX+PTX,(4)AuNR-lipid-DOX/PTX and(5)AuNR-lipid-DOX/PTX+NIR(0.5W/cm2,5min).Data were expressedas mean±SD(n=3)。
图8 AuNR-lipid-DOX/PTX体系的体内抗肿瘤结果;其中,(A)肿瘤相对生长曲线(数据归一化处理);(B)肿瘤照片;(C)各组肿瘤重量;(D)治疗期间荷瘤小鼠体重的变化;(E)不同组B16荷瘤小鼠肿瘤切片H&E染色图像(scale bar=50μm)。(1)对照组,(2)对照组+NIR,(3)AuNR,(4)AuNR+NIR,(5)lipid-DOX+PTX,(6)AuNR-lipid-DOX/PTX和(7)AuNR-lipid-DOX/PTX+NIR.**P≤0.01.n=5,mean±SD。
具体实施方式
本发明构建的AuNR-lipid-DOX/PTX纳米给药体系能够通过药物-光热-氧化应激的协同治疗作用,产生高效的抗肿瘤效果。该纳米平台的构建为线粒体靶向的多模式一体化治疗平台提供了新的策略。
在本申请中缩写物质名称对应如下:
实施例1纳米载体递送系统的制备
1)DSPE-PEG2000-DOX(lipid-DOX)的合成
将67.5mg DPSE-PEG2000-NHS和20mg DOX溶解10mL DMSO中,并加入12μL三乙胺,25℃搅拌24h后透析,每隔12h在透析袋中加入DMSO使样品完全溶解,重复6次后,再透析72h,将样品冻干后用1H-NMR确定结构。
2)金纳米棒(AuNR)的合成
将0.5mL CTAB(0.2M)溶液加入到0.5mL HAuCl4(0.5mM)溶液中搅拌均匀,然后滴加0.06mL冰冷的NaBH4(0.01M)溶液,至溶液变成棕黄色,继续搅拌2min,备用。在250mL的圆底烧瓶中加入50mLCTAB(0.2M)溶液,搅拌下加入2.8mL AgNO3(4mM)溶液、3.75mL HAuCl4(23mM)溶液以及47.5mL的超纯水,搅拌均匀。随后滴加1.6mL抗坏血酸(0.08M)溶液,在此过程中溶液逐渐变为无色。最后向体系中加入0.9mL的种子溶液,27-30℃避光搅拌15min,溶液变为紫红色,12000rpm离心10min,并用超纯水洗涤两次,定容25mL。
3)AuNR-lipid-DOX/PTX的合成
将lipid-DOX 2.0mg,DSPE-PEG2000-SH 0.185mg,CHOL 0.25mg,PTX 0.5mg溶解在10mL氯仿/甲醇混合溶液中(氯仿:甲醇=3:1),40℃旋转蒸发成膜,40℃空干燥过夜。用4mLAuNR(0.25mg/mL)溶液将薄膜超声溶解,搅拌24h后,超声波细胞粉碎机超声2min,14500rpm离心15min,超纯水洗涤一次,沉淀定容1mL。
实施例2理化性质表征
1)透射电子显微镜(TEM)
在具有碳支持膜的铜筛网上滴加AuNR以及AuNR-lipid-DOX/PTX溶液,待溶剂挥干后在TEM下观察其形态并采集图像,加速电压为120kV。结果如图1所示。从结果中可以看出,制备的金纳米棒分散性较好,长度大小为45~50nm(图1A),包裹了脂质层后(图1B),金纳米棒的形状并未发生改变。
2)粒径和Zeta电位的测定
采用DLS分析仪测定AuNR以及AuNR-lipid-DOX/PTX溶液的粒径和Zeta电位。将少量样品分散至超纯水中,超声分散均匀后进行测定,测试温度25℃。从图2A的结果可以看出,AuNR的平均粒径为139±3.5nm(PDI=0.351),粒径大于TEM的结果可能是由于AuNR表面的CTAB含有的基团彼此之间形成氢键所导致的;AuNR-lipid-DOX/PTX的平均粒径为188±3.3nm(PDI=0.267),粒径大于TEM的结果可能是由于被脂质层包裹后,整体分散性降低,导致测定结果偏大,而粒径的变化也间接说明了脂质层包裹的成功。AuNR和AuNR-lipid-DOX/PTX的Zeta电位分别为26±0.9mV和-26±3.4mV(图2B),电位的变化也间接说明了脂质层的成功包裹。
3)AuNR-lipid-DOX/PTX体系的光热曲线
主要考察了AuNR-lipid-DOX/PTX浓度、NIR照射强度对光热效果的影响,并考察了该体系的光热稳定性,结果如图3A和B所示。从图3A中可以看出,AuNR-lipid-DOX/PTX具有明显的剂量依赖性光热转换效率,体系浓度越大,光热效果越明显,体系的浓度100μg/mL时,NIR照射5min内温度就能升高25℃,显示出作为光热材料的巨大潜力。此外,如图3B所示,光热效果还表现出了NIR照射强度依赖性,体系的光热效果随着NIR照射强度的增加呈现上升的趋势。为了保证AuNR-lipid-DOX/PTX能够提供稳定光热效果,我们还考察了该体系的光热稳定性,从图3C中可以看出,在三次NIR照射循环中,光热曲线并没有发生明显的变化,说明该体系能够提供稳定的光热效果。
实施例3载药量的测定及药物体外释放
1)采用UV-vis和高效液相色谱(HPLC)分别测定AuNR-lipid-DOX/PTX中lipid-DOX和PTX的载药量。将制备好的AuNR-lipid-DOX/PTX用甲醇破坏溶解,离心去掉其中的AuNR,上清液分别用UV-vis和HPLC进行测定,采用外标法计算两种药物的含量。lipid-DOX的含量通过测定其和外标在UV-vis光谱中490nm处的吸光度,计算负载量。PTX的含量通过测定其和外标的在HPLC中的峰面积,计算负载量。HPLC测定条件为:吸收波长为227nm;流动相为甲醇:乙腈:水=40:40:20,流速为1mL/min。测得AuNR-lipid-DOX/PTX中lipid-DOX的负载量为20%(w/w,200mg/g),PTX的负载量为15%(w/w,150mg/g)。
2)在PBS 5.0+NIR的条件下测定了AuNR-lipid-DOX/PTX中lipid-DOX和PTX的释放曲线。即将AuNR-lipid-DOX/PTX分散在PBS 5.0的溶液中,在释放开始0-5min给予2.0W/cm2的NIR照射5min,将样品置于透析袋中,37℃条件下在恒温震荡箱中进行释放试验,并分别在0.5、1.0、2.0、4.0、8.0、12.0、24.0和48.0h取出500μL释放液,同时重新补加500μL PBS5.0。用荧光分光光度法测定释放液中lipid-DOX的浓度,并用HPLC测定释放液中PTX的浓度,最后计算两种药物的累积释放率。荧光分光光度计检测条件:激发波长为470nm,发射波长为554nm,扫描速度为240nm/min,扫描范围为480~700nm,狭缝宽度为10nm,电压为700V。结果如图4所示,药物的释放呈现了明显缓释的效果,而且lipid-DOX的释放比PTX快,这也间接说明了PTX被成功的包裹在了脂质层中。
实施例4细胞摄取及线粒体靶向实验
在铺有盖玻片的6孔板中按5×104/孔的细胞密度接种B16细胞并培养24h。细胞铺展后,加入作用浓度为50μg/mL的AuNR-lipid-DOX/PTX孵育2、4、6h。孵育结束后,加入线粒体染色剂Mito Tracker Green,37℃条件下孵育20min,PBS洗涤后用4%多聚甲醛溶液固定细胞20min,并用DAPI对细胞核染色20min,随后封片,用共聚焦显微镜对细胞摄取情况进行观察。从图5中可以看出,AuNR-lipid-DOX/PTX体系能被B16细胞成功摄取,并且该纳米体系可以明显的靶向至线粒体。和对照组相比,与纳米体系共孵育2h后,细胞内就能够观察到具有线粒体靶向性的lipid-DOX的红色荧光,而且与线粒体染色剂的绿色荧光具有很好的重叠性。随着时间的延长,进入细胞的AuNR-lipid-DOX/PTX越来越多,由此表明AuNR-lipid-DOX/PTX能有效地被内吞到肿瘤细胞内,并具有良好的线粒体靶向作用,这为该纳米体系在细胞内发挥作用奠定了基础。
实施例5体外细胞毒性实验
采用小鼠黑色素瘤B16细胞测定AuNR-lipid-DOX/PTX体系的细胞毒性。实验组分为AuNR、AuNR+NIR、lipid-DOX、PTX、lipid-DOX+PTX、AuNR-lipid-DOX、AuNR-PTX、AuNR-lipid-DOX/PTX和AuNR-lipid-DOX/PTX+NIR。在96孔板中按6×103/孔的细胞密度接种B16细胞并孵育24h。将不同浓度的以上体系加入到96孔板中,每组AuNR作用浓度均分别为1.0、5.0、25.0、50.0、100.0μg/mL,NIR组每孔均给予0.5W/cm2的NIR(808nm)照射5min,之后再孵育20h。孵育结束后,在各孔中加入20μL的MTT(5mg/mL)溶液,再孵育4h。弃掉培养基后,每孔均加入200μL的DMSO并混匀。用酶标仪测定各孔在570nm处的吸光度值,并以630nm作为参比。结果如图6所示,AuNR本身并没有明显的细胞毒性,在浓度达到100μg/mL时,细胞存活率还在90%以上,而在给予0.5W/cm2的NIR照射5min后,细胞存活率明显下降,在100μg/mL时,细胞存活率仅为36%,由此可以说明AuNR具有很好的光热治疗效果。而且除了AuNR组,其余各组均表现出了不同程度的细胞杀伤能力。AuNR-lipid-DOX/PTX的细胞抑制率与lipid-DOX+PTX组存在显著性差异(P<0.05),这是由于一体化纳米平台构建成功后,整个体系均被lipid-DOX靶向至线粒体,lipid-DOX和PTX两者共同作用于线粒体,诱导细胞凋亡并刺激线粒体产生强烈的氧化应激所引起的。值得关注的是,AuNR-lipid-DOX/PTX+NIR组的细胞杀伤作用明显优于其他组,这是由于光热治疗的参与不仅能通过热消融杀死肿瘤细胞,还能够刺激细胞产生氧化应激,最终实现了线粒体靶向性的药物、光热和氧化应激效应,实现了高效的细胞杀伤效果。
实施例6细胞内ROS的产生
采用共聚焦显微镜时,在铺有盖玻片的6孔板中按8×104/孔的细胞密度接种B16细胞并培养24h。细胞铺展后,分别加入PBS、AuNR-PTX、AuNR-lipid-DOX、lipid-DOX+PTX,AuNR-lipid-DOX/PTX以及AuNR-lipid-DOX/PTX+NIR孵育4h,作用浓度按照AuNR 50μg/mL计算,其中NIR组在孵育结束后给予0.5W/cm2的NIR照射5min。随后用PBS洗涤,并加入DCFH-DA在37℃下孵育20min,PBS洗涤后用4%多聚甲醛溶液固定细胞20min,并用DAPI对细胞核染色20min,随后封片,并用共聚焦显微镜进行观察。结果如图7所示,与对照组相比,AuNR-PTX组能够产生的少量的ROS;而AuNR-lipid-DOX组相比于对照组ROS荧光强度增加了1.65倍,这可能是由于线粒体靶向作用导致氧化应激作用增强,ROS产生增多;AuNR-lipid-DOX/PTX产生的ROS多于lipid-DOX+PTX,这应该是由于PTX也被靶向至线粒体,导致整个体系产生的氧化应激增强,ROS产生增多;AuNR-lipid-DOX/PTX+NIR产生的ROS为对照组的2.2倍,明显高于其他组,这应该是由于光热也刺激细胞发生氧化应激,药物、光热协同作用,导致ROS产生明显增强。
实施例7体内抗肿瘤效果的评价
1)小鼠B16黑色素瘤肿瘤模型的建立
动物实验按河北医科大学科研管理处批准的方案进行。富集培养的B16细胞并分散在4mL的PBS中,使其细胞密度为1×107个/mL。取35只C57小鼠,分别将100μL的B16细胞PBS悬液通过皮下注射的方式注射到小鼠的前肢后部。
2)实验分组与给药方案
当肿瘤体积达到约100mm3时,随机将35只荷瘤的C57小鼠分为7组:PBS、PBS+NIR、AuNR、AuNR+NIR、lipid-DOX+PTX、AuNR-lipid-DOX/PTX和AuNR-lipid-DOX/PTX+NIR组,第0天采用瘤内注射的方式对各组小鼠进行给药,给药量为100μL/只,每组相应的AuNR作用浓度均为100μg/mL,其中所有NIR组在给药后4h给予0.5W/cm2的NIR(808nm)照射5min。每天测量小鼠的体重和肿瘤体积并记录,肿瘤体积通过下述公式进行计算:
Tumor volume(mm3)=(l×w2)/2其中l=肿瘤长度,w=肿瘤宽度。
结果如图8所示,每天测量肿瘤体积(图8A),10天后收集肿瘤并称重(图8B和C)。如图8所示,PBS组、PBS+NIR组和AuNR组对肿瘤生长均没有抑制作用,表明单纯的NIR照射和AuNR对肿瘤没有治疗效果;与PBS组相比,AuNR+NIR,lipid-DOX+PTX,AuNR-lipid-DOX/PTX和AuNR-lipid-DOX/PTX+NIR组肿瘤的生长均受到不同程度的抑制。其中AuNR-lipid-DOX/PTX组的抑瘤效果明显好于lipid-DOX+PTX组(P<0.01),这一方面是由于AuNR-lipid-DOX/PTX具有药物缓释作用,实现了长效治疗,另一方面是由于PTX也被到了线粒体,提高了治疗效果;相比于AuNR-lipid-DOX/PTX组,AuNR-lipid-DOX/PTX+NIR组表现出更加优越的抗肿瘤效果(P<0.01),由此说明药物、光热和氧化应激协同作用能起到更好的治疗效果。
此外,在治疗后10天内,各给药组小鼠的体重与对照组相比无明显差异(图8D),表明AuNR-lipid-DOX/PTX具有良好的生物安全性,对小鼠其他器官组织无明显副作用。对各组的肿瘤进行H&E染色,结果如图8E所示,与对照组、单纯NIR组以及AuNR组相比,其他组均可观察到坏死或凋亡区域。尤其是AuNR-lipid-DOX/PTX+NIR组肿瘤切片出现密集、大面积的坏死或凋亡,显示出最好的杀死肿瘤细胞的作用。
Claims (10)
1.一种线粒体靶向的光热/化疗协同的纳米药物递送粒子,其特征在于所述纳米药物递送粒子为以金纳米棒为核,以阿霉素脂质体为壳的核壳结构,其粒径长度为45~50nm,宽度为12~14nm。
2.根据权利要求1所述的纳米药物递送粒子,其特征在于,所述核壳结构通过金-硫键及脂质体互插作用构建。
3.根据权利要求1所述的纳米药物递送粒子,其特征在于还包括负载于所述脂质体层的纳米药物紫杉醇。
4.根据权利要求2要求所述的纳米载体递送粒子,其特征在于所述紫杉醇的包封率为15%~20%。
5.一种如权利3或4所要求的纳米药物递送粒子的制备方法,其特征在于,包括以下步骤:
1)将DPSE-PEG2000-NHS和DOX溶解DMSO中,并加入三乙胺,搅拌后透析,得到lipid-DOX;
2)种子溶液制备;将CTAB溶液加入到HAuCl4溶液中搅拌均匀,然后滴加冰冷的NaBH4溶液,至溶液变成棕黄色,继续搅拌,得到种子溶液备用;
AuNR溶液制备:在圆底烧瓶中加入CTAB溶液,搅拌下加入AgNO3溶液、HAuCl4溶液以及超纯水,搅拌均匀,随后滴加抗坏血酸溶液,在此过程中溶液逐渐变为无色,最后向体系中加入所述种子溶液,避光搅拌,溶液变为紫红色,离心,并用超纯水洗涤,加水制备成AuNR溶液;
3)将所述步骤1)得到的产物,DSPE-PEG2000-SH、CHOL、PTX溶解在氯仿/甲醇混合溶液中,旋转蒸发成薄膜,40℃真空干燥过夜,用所述步骤2)得到的溶液将所述薄膜超声溶解,搅拌后,超纯水洗涤一次,得到产物。
6.根据权利要求5所述的制备方法,其特征在于所述步骤1)中,所述DPSE-PEG2000-NHS和所述DOX的质量比为67.5:20。
7.根据权利要求5所述的制备方法,其特征在于所述步骤2)中,所述种子溶液制备步骤中所述CTAB和所述HAuCl4和所述NaBH4的摩尔比为2:0.005:0.1;所述AuNR溶液制备中,所述HAuCl4与所述CTAB和和AgNO3的摩尔比为23:20:4;所述HAuCl4与所述种子的摩尔比为46:1。
8.根据权利要求5所述的制备方法,其特征在于所述步骤3)中,所述lipid-DOX、SPE-PEG2000-SH、CHOL和PTX和所述AuNR的质量比为2.0:0.185:0.25:0.5:1。
9.根据权利要求5所述的制备方法,其特征在于所述纳米载体递送粒子中PTX负载率为15wt%,DOX负载率为20wt%。
10.权利要求1-4任一项所述的纳米药物递送粒子在制备黑色素瘤化疗药物、光热联合治疗药物方面的应用。
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